WO2003054232A2 - Procedes de suppression des oligonucleotides bicatenaires contenant des erreurs de sequençage a l'aide de proteines de reconnaissance de mesappariements - Google Patents

Procedes de suppression des oligonucleotides bicatenaires contenant des erreurs de sequençage a l'aide de proteines de reconnaissance de mesappariements Download PDF

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WO2003054232A2
WO2003054232A2 PCT/US2002/040138 US0240138W WO03054232A2 WO 2003054232 A2 WO2003054232 A2 WO 2003054232A2 US 0240138 W US0240138 W US 0240138W WO 03054232 A2 WO03054232 A2 WO 03054232A2
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double
dna
oligonucleotides
stranded oligonucleotides
stranded
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PCT/US2002/040138
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WO2003054232A3 (fr
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John C. Tabone
John T. Mulligan
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Blue Heron Biotechnology, Inc.
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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

Definitions

  • the present invention in certain embodiments is directed toward the removal of double-stranded oligonucleotides containing sequence errors. It is more particularly related to the removal of error-containing oligonucleotides (such as error- containing double-stranded DNA), generated for example by chemical or enzymatic synthesis (including by PCR amplification), by removal of mismatched duplexes using mismatch recognition proteins.
  • error-containing oligonucleotides such as error- containing double-stranded DNA
  • DNA is used as a prototypical example of an oligonucleotide. Mismatches are formed directly during chemical DNA synthesis or are formed in enzymatically synthesized DNA by denaturing and reannealing a mixed population of correct and error-containing DNA.
  • oligos oligonucleotides
  • oligos are used as building blocks for DNA synthesis and are synthesized as single strands using automated oligonucleotide synthesizers. Random chemical side reactions create base errors in these single-stranded oligos.
  • two complementary synthetic oligos are hybridized to form double-stranded DNA, there is almost no chance that the random base errors formed in one strand will be correctly base paired in the opposite strand. It is these incorrectly paired bases that form the mismatches found in chemically synthesized double-stranded DNA.
  • an enzyme such as a polymerase
  • This template contains the same type of base mismatches that are found in the synthetic DNA described above.
  • the mismatches are converted into base paired errors in sequence.
  • These base pairings of the mismatches occur as polymerase synthesizes the complementary base on the strand opposing strand.
  • the result of this enzymatic step is to create a mixed population of DNA molecules where all bases are paired correctly with both correct (error- free) and incorrect (error-containing) sequences.
  • the polymerase step essentially maintains the ratio of correct to incorrect sequence.
  • a DNA population such as that formed from enzymatic DNA synthesis containing both error-free and error-containing base paired DNA where both are correctly base pair matched can be converted to a population composed of both mismatched and error-free correctly base paired DNA by denaturation and reannealing.
  • these steps are performed on a population that contains a small fraction of error-containing molecules relative to correct molecules, the vast majority of error containing strands will hybridize with the more abundant correct strand and will form mismatched sites.
  • the present invention provides a variety of methods for removing double-stranded oligonucleotide (e.g. , DNA) molecules containing one or more sequence errors generated during nucleic acid synthesis, from a population of correct oligonucleotide duplexes.
  • the oligonucleotides are generated enzymatically .
  • Heteroduplex oligonucleotides may be created by denaturing and reannealing the population of duplexes. The reannealed oligonucleotide duplexes are contacted with a mismatch recognition protein that interacts with the duplexes contaimng a base pair mismatch.
  • oligonucleotide heteroduplexes that have interacted with the protein are separated from homoduplexes as the latter do not interact with the protein. These methods are also used to remove heteroduplex oligonucleotides (e.g. , DNA) that are formed directly from chemical nucleic acid synthesis.
  • heteroduplex oligonucleotides e.g. , DNA
  • the present invention provides a method of depleting in a sample of double-stranded oligonucleotides a population of double-stranded oligonucleotides containing mismatched bases thereby enriching in said sample a population of double-stranded oligonucleotides containing correctly matched bases, comprising the steps of: (a) contacting said sample containing double-stranded oligonucleotides with a mismatch recognition protein under conditions to permit the protein to interact with a double-stranded oligonucleotide containing at least one mismatched base; and (b) collecting double-stranded oligonucleotides that have not interacted with said mismatch recognition protein, thereby depleting the population of double-stranded oligonucleotides containing mismatched bases.
  • an additional step comprising separating said double-stranded oligonucleotide containing at least one mismatched base that has interacted with said mismatch recognition protein, from double-stranded oligonucleotides that have not interacted with said mismatch recognition protein.
  • RNA Natural bases of DNA - adenine (A), guanine (G), cytosine (C) and thymine (T). In RNA, thymine is replaced by uracil (U).
  • Synthetic double-stranded oligonucleotides - two strands of oligonucleotides (e.g., substantially double-stranded DNA) composed of single strands of oligonucleotides synthetically produced (e.g. , by chemical synthesis or by the ligation of synthetic double-stranded oligonucleotides to other synthetic double-stranded oligonucleotides to form larger synthetic double-stranded oligonucleotides) and joined together in the form of a duplex.
  • synthetic double-stranded oligonucleotides composed of single strands of oligonucleotides synthetically produced (e.g. , by chemical synthesis or by the ligation of synthetic double-stranded oligonucleotides to other synthetic double-stranded oligonucleotides to form larger synthetic double-stranded oligonucleotides) and joined together in the form of a duplex.
  • Synthetic failures - undesired products of oligonucleotide synthesis such as side products, truncated products or products from incorrect ligation.
  • Mismatch recognition protein a protein that recognizes heteroduplex oligonucleotides (e.g., heteroduplex DNA); typically the protein is a mismatch repair enzyme or other oligonucleotide binding protein (e.g., DNA mismatch repair enzyme or other DNA binding protein); the protein may be isolated or prepared synthetically (e.g., chemically or enzymatically), and may be a derivative, variant or analog, including a functionally equivalent molecule which is partially or completely devoid of amino acids.
  • the present invention is directed in certain embodiments toward methods for the removal of error-containing double-stranded oligonucleotide (e.g. , DNA) molecules from a population of double-stranded oligonucleotides (e.g.
  • the error-containing oligonucleotide molecules in this population are removed from the correct molecules when the errors are present as mismatches in the double-stranded oligonucleotides.
  • the removal of the mismatch is based in the present invention on the use of mismatch recognition proteins that recognize mismatched bases in double-stranded oligonucleotides. Such proteins interact with double-stranded oligonucleotides containing mismatched bases (e.g. , by binding and/or cleaving on or near the mismatch site).
  • the protein step may or may not be performed in conjunction with a separation step (e.g., chromatographic step) to separate mismatch-containing heteroduplex from homoduplex oligonucleotides.
  • a separation step e.g., chromatographic step
  • mismatch recognition proteins may be used to deplete an oligonucleotide population of those double-stranded oligonucleotides which contain sequence errors.
  • Depletion of error-containing oligonucleotides from the desired double-stranded oligonucleotides refers generally to at least about (wherein "about” is within 10%) a two-fold depletion relative to the total population prior to separation. Typically, the depletion will be a change of about two-fold to three-fold from the original state.
  • the particular fold depletion may be the result of a single use of the method (e.g., single separation) or the cumulative result of a plurality of use (e.g., two or more separations).
  • Depletion of error-containing oligonucleotides is useful, for example, where the oligonucleotides are double-stranded DNA which correspond to a gene or fragments of a gene.
  • Oligonucleotides suitable for use in the present invention are any double-stranded sequence. Examples of such oligonucleotides include double-stranded DNA, double-stranded RNA, DNA/RNA hybrids, and functional equivalents containing one or more non-natural bases.
  • Preferred oligonucleotides are double-stranded DNA.
  • Double-stranded DNA includes full length genes and fragments of full length genes. For example, the DNA fragments may be portions of a gene that when joined form a larger portion of the gene or the entire gene.
  • the present invention provides a preparative method to remove base mismatched oligonucleotides from a population of correctly base matched oligonucleotides.
  • the method generally comprises the steps of contacting a double- stranded oligonucleotide sample with a mismatch recognition protein, and collecting the double-stranded oligonucleotides that have not interacted with the mismatch recognition protein. Collecting the double-stranded oligonucleotides that have not interacted with the protein can be the result of their removal from the sample, or the removal from the sample of those oligonucleotides that did interact.
  • the step of contacting is performed under conditions (including a time sufficient) to permit a mismatch recognition protein to interact with (e.g. , bind to and/or cleave) mismatch-containing heteroduplex oligonucleotides.
  • the method may, prior to the step of collecting, optionally include a step of separating the double-stranded oligonucleotide that contains at least one (one or more) mismatched base and that has interacted with the mismatch recognition protein, from double-stranded oligonucleotides that have not interacted with the mismatch recognition protein.
  • the method may, in place of or in addition to a separation step and prior to the step of contacting, optionally include steps of first denaturing and then reannealing a sample of double-stranded oligonucleotides under conditions to permit conversion of the double- stranded oligonucleotides first to single-stranded oligonucleotides and then to double- stranded oligonucleotides. It will be evident to one of ordinary skill in the art that the steps may be performed sequentially, or two or more steps may be performed simultaneously. For example, in an embodiment where a mismatch recognition protein is immobilized on a solid support, the step of contacting results directly in separation.
  • the mismatch recognition proteins share the property of binding on or within the vicinity of a mismatch.
  • a protein reagent includes proteins that are endonucleases, restriction enzymes, ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases and antibodies specific for mismatches. Variants of these proteins can be produced, for example, by site directed mutagenesis, provided that they are functionally equivalent for mismatch recognition.
  • the enzyme can be selected, for example, from T4 endonuclease 7, T7 endonuclease 1, SI, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavase, and HINFl .
  • the mismatch recognition protein cleaves at least one strand of the mismatched DNA in the vicinity of the mismatch site.
  • the optional separation step can be performed in a variety of means, e.g., using high performance liquid chromatography (HPLC), by size exclusion chromatography, ion exchange chromatography, affinity chromatography or reverse phase chromatography.
  • HPLC high performance liquid chromatography
  • the separation can also be performed using membranes in a slot blot fashion or a microtiter filter plate.
  • the separation may also be performed using solid phase extraction cartridges using supports similar to the HPLC columns.
  • a mismatch recognition protein (e.g. , the MutS protein from E. coli) is immobilized on a solid support.
  • Methods for immobilizing proteins on solid supports are well known to one in the art, and include covalent or noncovalent attachment to a solid support.
  • types of suitable solid supports are well known to one in the art, and include beads, glass, polymers, resins and gels. The following is a representative example for preparing oligonucleotides depleted of error-containing oligonucleotides.
  • Two complementary oligonucleotides e.g., DNA
  • duplex oligonucleotides e.g., double-stranded DNA.
  • double-stranded DNA may be enzymatically synthesized (and further denatured and reannealed).
  • This mixture is passed over a column with a mismatch recognition protein (e.g. , the MutS protein) immobilized on a solid support (such as beads) in the column.
  • a mismatch recognition protein e.g. , the MutS protein
  • Fragments with an error in either of the oligonucleotides will usually contain a mismatch since in most cases the other strand is correct at that position.
  • Duplexes containing mismatches will bind to the column and only error-free duplexes will be enriched in the flow-through from the column.
  • a gene encoding a mismatch recognition protein (e.g., the MutS gene) is fused to a gene fragment that encodes a binding domain (for instance a chitin-binding domain).
  • a binding domain for instance a chitin-binding domain.
  • the fused protein is produced and mixed with a duplex fragment that is produced as described above. Duplex molecules with an error in either strand will bind to the fusion protein (e.g. , MutS fusion protein). After an appropriate incubation, the mixture is passed over a chitin column. The fusion protein binds to the column via the chitin. Duplex molecules with mismatches are retained on the column, and error-free duplexes flow through.
  • Beta-galactosidase is an enzyme that can convert X-gal from a colorless compound into a brilliant blue compound (Marmiatis; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989).
  • the lad gene encodes a repressor of beta-galactosidase synthesis in E. coli. In a cell with functional lac repressor, the synthesis of beta-galactosidase is suppressed and colonies grown on X-gal plates are white. If the lac repressor gene is inactive, beta-galactosidase is produced and the colonies are a bright blue color.
  • a 205 base pair segment of the lad gene with the sequence:
  • oligonucleotides used to make the gene are prepared using an Oligo
  • oligonucleotides are HPLC-grade acetonitrile and water obtained from Burdick & Jackson (Muskegon, MI). Triethylarnmonium acetate (TEAA), pH 7.0, and 3% Trifluoroacetic acid in water are obtained from Glen Research. After purification, the synthesized oligonucleotides are evaporated to dryness in a SpeedVac (Savant, Farmingdale, NY) and resuspended in HPLC grade water. Concentrations of the oligonucleotides are determined by reading the 260 nm absorbance on a Pharmacia LKB Ultrospec III (Amersham Pharmacia, Upsala, Sweden).
  • the oligonucleotides are used to form duplex fragments by drying 500 pmoles each of the complementary oligonucleotides in a speedvac and resuspending in 10 microliters TE. A 5 microliter sample of the solution (250 pmoles) is mixed with 10 microliters of 2XSSPE (prepared according to Manniatis), heated to 95°C and cooled to room temperature.
  • 2XSSPE prepared according to Manniatis
  • Duplexes are successively ligated together to make longer fragments until the full length product is made.
  • Each ligation consists of 500 picomoles of a pair of double-stranded oligonucleotide, 3 microliters of 10X ligation buffer (Fermentas Inc., Hanover, MD), 10 units of T4 DNA ligase (product # EL0016, Fermentas) and water to make a total volume of 30 microliters. All duplexes are ligated together under the same conditions. Each ligation mix is incubated at 37°C for 60 minutes, heated to 65 °C for 10 minutes and the fragment isolated by HPLC.
  • HPLC High performance liquid chromatography
  • Varian Inc. Winut Creek, CA
  • HPLC High performance liquid chromatography
  • the column used is a Zorbax Eclipse dsD ⁇ A Analysis Column (4.6 mm ID x 75 mm, 3.5 micron) equipped with an in line Guard Column (4.6 mm ID x 12.5 mm, 3.5 micron) both from Agilent Technologies, Inc.
  • the synthetic fragment produced in Example 1 is cloned into the lad gene to test its function.
  • Three micrograms of plasmid vector pWBlOOO (Lehming et al., Proc. Natl. Acad. Sci. USA, 85:7947-7951, 1988) is digested with restriction enzymes EcoRl and Hindlll and the vector fragment gel purified using a Strata Prep DNA extraction kit (Stratagene product #400766) according to the manufacturers instructions, and resuspended in 100 microliters of TE.
  • One microgram of the la fragment is treated with T4 polynucleotide kinase, extracted once with phenol and once with chloroform, ethanol precipitated and resuspended in 20 microliters of TE.
  • Five microliters of the cut vector and one microliter of the synthetic lad fragment are ligated in a total volume of 100 microliters using Fermentas T4 DNA ligase according to the manufacturers instructions.
  • the ligation mix is extracted once with Strataclean, concentrated and washed twice with 1/10 concentration TE and brought to a volume of 10 microliters in 1/10 th concentration TE.
  • One microliter of this mix is transferred into E.
  • coli strain DC 41-2 carrying plasmid pWB310 (Lehming et al., EMBO 6:3145-3153, 1987) by electroporation using a BTX ECM399 electroporator (Genetronics, Inc., San Diego, CA) according to the manufacturers instructions. Colonies are grown overnight on LB plates in the presence of 10 mg/liter tetracycline, 200 mg/liter ampicillin, 60 mg/liter X-gal and 300 mg/liter IPTG. Colonies carrying a plasmid with a functional lad gene are white; those without a functional lad gene are blue.
  • DIAMINOPURINE AT BASES 86, 88, 133, OR 178 One common side reaction of oligonucleotide synthesis is the formation of diaminopurine from a dG residue in the DNA chain. Modified oligonucleotides containing 2,6-diaminopurine are obtained from Trilink Biotechnologies (San Diego, CA) and incorporated into the 205 bp lad gene fragment. Four samples are prepared as described. in Example 1 , with one diaminopurine residue (labeled D below) substituted for a dG residue in each sample. Oligonucleotide Fragment Name Base Replaced 5 ' ACCGTTTCTADAGTGGTTAACCAGG 3 ' D-T86 86
  • a second common side reaction of oligonucleotide synthesis is deamination of the N4-amine of deoxycytidine to form a uracil (dU) in the DNA chain.
  • Modified oligonucleotides containing uracil (dU) are obtained from Midland Certified Reagent Company (Midland, TX) and incorporated into the 205 bp lad gene fragment. Two samples are prepared as described in Example 1, with one uracil residue (labeled dU below) substituted for a dC residue in each sample.
  • a third common side reaction of oligonucleotide synthesis is the formation of abasic sites by depurination of protected adenosine residues during chain elongation.
  • Modified oligonucleotides containing uracil are obtained from Midland Certified Reagent Company (Midland, TX) and incorporated into the 205 bp lad gene fragment. Two samples are prepared as described in Example 1, with one uracil residue (labeled dU below) substituted for a dA residue in each sample.
  • the DNA is treated with Uracil-N-Glycosylase (Epicentre Technologies Corp., Madison, WI) according to the manufacturers instructions to remove the uracil base, leaving an apurinic site in place of the corresponding A residue in the native 205 base pair fragment.
  • Uracil-N-Glycosylase Epicentre Technologies Corp., Madison, WI
  • the thermal and gradient conditions for isolating chemically-pure enriched sequence are calculated using the DHPLC Melt Program (http://insertion.stanford.edu/melt.html) available from Stanford University (Palo Alto, CA) and available for license from the Stanford University Office of Technology Licensing referring to the docket number S95-024.
  • the 4 base single-stranded region on either end of the 205 base pair fragment is removed to give the following 197 base pair sequence.
  • the gradients are specified below as percent buffer B at times 1, 2 and 3 (Bl, B2, B3).
  • the gradient is run from Bl to B2 in 0.5 minutes, then B2 to B3 in 3.0 minutes.
  • the chromatographic behavior of the native lad DNA and the eight modified lad DNA are measured in response to a range of gradient and temperature conditions.
  • the lad DNA is below:
  • the fragments produced in Example 3, Example 4 and Example 5 are amplified using PCR to convert the base pair mismatches in the synthetic fragments into base paired errors in the enzymatically produced fragments.
  • the PCR is designed to amplify the complete fragment and add sequence using tails on the primers to add cloning sites for EcoRl and Hindlll restriction enzymes.
  • Reverse primer 5 ' -CTTCGGAAGATCCTTAGCTTTGTTTACCAGCCAGCTG-3 '
  • Example 4 and Example 5 are described in the table below. All the components are combined and vortexed to ensure good mixing, and centrifuged. Aliquots are then distributed into PCR tubes as shown in the following table:
  • Pfu 1 OX Buffer (Cat. No.600153-82, Stratagene, Inc., La Jolla, CA) 5 ⁇ L lO mM dNTP Mix 1 ⁇ L
  • Reverse primer (lO pmol/ ⁇ L) 1 ⁇ L
  • PFUTurboTM (Cat. No. 600250, Stratagene, Inc., La Jolla, CA) 1 ⁇ L
  • thermocycler MJ Instruments
  • PCR products are purified using the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA) according to the manufacturers instructions and resuspended in 10 microliters of TE.
  • Example 8 The enzymatic fragments produced in Example 8 are cloned into the lad gene to test their biological function.
  • Ten micrograms of plasmid vector pWBlOOO (Lehming et al., PNAS 85 :7947-7951 , 1988) and each of the PCR reactions from Example 8 is digested with restriction enzymes EcoRl and Hindlll .
  • Each of the cut amplification products and the vector fragment are gel purified using a Strata Prep DNA extraction kit (Stratagene product #400766) according to the manufacturers instructions, and resuspended in 100 microliters of TE.
  • Each of the cut PCR reactions and one microgram of each lad fragment is treated with T4 polynucleotide kinase, extracted once with phenol and once with chloroform, ethanol precipitated and resuspended in 20 microliters of TE.
  • Five microliters of the cut vector and the entire sample of the amplified DNA are ligated in a total volume of 100 microliters using New England Biolabs T4 DNA ligase according to the manufacturers instructions.
  • the ligation mix is extracted once with Strataclean, concentrated and washed twice with 1/10 concentration TE and brought to a volume of 10 microliters in 1/10 concentration TE. One microliter of this mix is transferred into E.
  • coli strain DC 41 -2 carrying plasmid pWB310 (Lehming et al., EMBO 6:3145-3153, 1987) by electroporation using a BTX ECM399 electroporator according to the manufacturers instructions. Colonies are grown overnight on LB plates in the presence of 10 mg/liter tetracycline, 200 mg/liter ampicillin, 60 mg/liter X-gal and 300 mg/liter IPTG. Colonies carrying a plasmid with a functional lad gene are white; those without a functional lad gene are blue. Each modified fragment is characterized by the frequency of blue colonies relative to the frequency of blue colonies derived from clones of the native synthetic lad fragment as described in Example 2.
  • the ability of the chromatographic technique to enrich a population of enzymatically amplified base paired DNA composed of "correct" DNA in the presence of "incorrect” DNA is shown by spiking native lad DNA with each of the eight amplified la DNA from Example 8, denaturing and reannealing the mix, and enriching for the correct DNA using HPLC.
  • Example 7 The remainder of each of these samples is chromatographed using thermal and gradient conditions (identified in Example 7) which alter the mobility of the modified fragments relative to the native fragment. For each sample, the peaks are collected with a fraction collector as described in Example 1 at the elution time determined in Example 7. Two fractions are collected, one with a mobility characteristic of the modified DNA fragments and one with a slower mobility characteristic of the native DNA fragment. These fractions are dried down and cloned as described in Example 9. In parallel, a portion of each of the eight unfractionated mixtures is cloned and tested in the same way. The "native fraction" fragments show a lower number of sequence errors than the original mixtures or the early-eluting fractions, as indicated by the frequency of blue colonies.
  • mismatch binding protein to enrich a population of enzymatically amplified base paired DNA composed of "correct" DNA in the presence of "incorrect” DNA is shown by spiking native lad DNA with each of the eight amplified lad DNA from Example 8, denaturing and reannealing the mix, and enriching for the correct DNA using mismatch binding protein immobilized to magnetic beads.
  • an equimolar mixture is prepared of amplified native and amplified fragments by mixing 20 pmoles of the amplified DNA with 20 pmoles of the amplified native fragment. A fraction of each mixture is retained for functional testing as described below.
  • EXAMPLE 12 ENRICHMENT OF NATIVE LACI FRAGMENTS FROM MIXTURES OF NATIVE AND ENZYMATICALLY AMPLIFIED FRAGMENTS OF THE LACI GENE CARRYING MODIFIED BASES BY REMOVAL OF MISMATCHES WITH A MISMATCH BINDING PROTEIN PASSED
  • mismatch binding protein to enrich a population of enzymatically amplified base paired DNA composed of "correct" DNA in the presence of "incorrect” DNA is shown by spiking native la DNA with each of the eight amplified la DNA from Example 8, denaturing and reannealing the mix, and enriching for the correct DNA using mismatch binding protein passed through a nitrocellulose filter.
  • an equimolar mixture is prepared of amplified native and amplified fragments by mixing 20 pmoles of the amplified DNA with 20 pmoles of the amplified native fragment. A fraction of each mixture is retained for functional testing as described below.

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Abstract

Dans certains modes de réalisation, l'invention concerne des procédés de suppression des molécules d'oligonucléotides bicaténaires (par ex., l'ADN) qui contiennent une ou plusieurs erreurs de séquençage produites au cours de la synthèse des acides nucléiques, dans une population de duplex oligonucléotidiques corrects. Dans un mode de réalisation, ces oligonucléotides sont obtenus par voie enzymatique. Pour obtenir des oligonucléotides hétéroduplex (qui contiennent des bases mésappariées), la population des duplex est soumise à une dénaturation puis à une renaturation, par exemple. Les duplex oligonucléotidiques soumis à la renaturation sont mis en contact avec une protéine de reconnaissance de mésappariement qui interagit (par fixation et/ou clivage) avec les duplex qui présentent un mésappariement dans les paires de bases. Les hétéroduplex oligonucléotidiques ayant interagi avec une telle protéine sont séparés des homoduplex (simultanément au contact avec les homoduplex, ou séquentiellement, dans une autre opération). Ces procédés sont également utilisés dans un autre mode de réalisation pour supprimer les oligonucléotides hétéroduplex (par ex. l'ADN) qui sont formés directement par la synthèse chimique des acides nucléiques.
PCT/US2002/040138 2001-12-13 2002-12-12 Procedes de suppression des oligonucleotides bicatenaires contenant des erreurs de sequençage a l'aide de proteines de reconnaissance de mesappariements WO2003054232A2 (fr)

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WO2006026614A2 (fr) * 2004-08-27 2006-03-09 Wisconsin Alumni Research Foundation Procédé de réduction d'erreur dans des populations d'acides nucléiques
EP2246417A1 (fr) 2004-04-22 2010-11-03 Talecris Biotherapeutics, Inc. Plasmine modifiee par recombinaison
US7879580B2 (en) 2002-12-10 2011-02-01 Massachusetts Institute Of Technology Methods for high fidelity production of long nucleic acid molecules
US7932025B2 (en) 2002-12-10 2011-04-26 Massachusetts Institute Of Technology Methods for high fidelity production of long nucleic acid molecules with error control
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