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WO2000046408A1 - Procede de replication d'adn - Google Patents

Procede de replication d'adn Download PDF

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Publication number
WO2000046408A1
WO2000046408A1 PCT/US2000/004445 US0004445W WO0046408A1 WO 2000046408 A1 WO2000046408 A1 WO 2000046408A1 US 0004445 W US0004445 W US 0004445W WO 0046408 A1 WO0046408 A1 WO 0046408A1
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Prior art keywords
dna
loop
replication
target
replisome
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PCT/US2000/004445
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English (en)
Inventor
Kenneth Marians
Liu Joing
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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Priority to US09/890,829 priority Critical patent/US6699693B1/en
Publication of WO2000046408A1 publication Critical patent/WO2000046408A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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

Definitions

  • This application relates to a process for DNA replication, and to the application of this process for a variety of purposes.
  • Replication of DNA and other nucleic acids is a complex natural phenomenon which occurs within all biological systems.
  • To facilitate the exploitation of the resources represented in the diverse genetic materials of the world's organisms it is desirable to be able to replicate selected DNA sequences under more controlled conditions, for example to produce increased amounts of one sequence.
  • Such replication of selected DNA sequences is required for a great many applications of potential scientific and industrial significance, and has been accomplished by a variety of techniques. These include cloning of the DNA sequences into plasmids or genes, and replication of the plasmid using the DNA replication mechanisms of a host organism, and amplification techniques such as PCR or ligase amplification.
  • Cloning is capable of replicating complete gene sequences, but requires the introduction of the sequences into a host organism, and the subsequent recovery of the duplicated DNA.
  • PCR and similar amplification techniques offer increased flexibility, including the ability to introduce labels and/or sequence variations into the replicated DNA, and avoid the use of a host organism, but are limited in the length of the sequence which can be replicated.
  • a methodology which will permit the replication of long DNA molecules, while providing the flexibility associated with PCR amplification. It is an object of the present invention to provide such a methodology. Summary of the Invention
  • the present invention provides a method for replicating DNA, and in particular for replicating large segments of DNA.
  • a primer is combined with a target DNA molecule to be replicated.
  • the primer is designed to be at least partially homologous to a known site on the target DNA, and to create a D-loop when hybridized with that site.
  • a replisome is then assembled at the D-loop, and this replisome creates a copy of the DNA, starting at the primer binding site.
  • Fig. 1 shows the scheme used for making a double- stranded circular template
  • DNA molecule containing a D-loop which was used to validate the concept of the invention.
  • the present invention provides a method for the controlled replication, generally in vitro, of selected regions of DNA.
  • replication of a target region of a target DNA molecule is accomplished by:
  • ATP is preferably provided at concentrations in excess of about 1 mM.
  • ATP is required because the formation of a processive DNA polymerase complex requires
  • D-loop at a selected initiation site in duplex DNA can be accomplished using an oligonucleotide primer which hybridizes with double- stranded DNA at a selected initiation site. The non-hybridized strand is displaced to create the D-loop. D- loop formation can be driven by the homologous pairing enzyme, RecA, as has been described in the literature. See, McEntee et al., Proc. Nat'l Acad. Sci. (USA) 76: 2615-2619
  • D-loop formation could also be driven by other methods, for example heating at a moderately high temperature (for example 75- 80°C) may be enough to drive annealing, particularly in regions rich in A+T bases.
  • a moderately high temperature for example 75- 80°C
  • the oligonucleotide primer which is used for generation of the D-loop generally has a length of from 20 to about 50 bases.
  • the primer is selected to be substantially complementary to one of the two strands of the target DNA duplex at the initiation site.
  • substantially complementary refers to a primer which will hybridize with the target DNA duplex under conditions of moderately high stringency.
  • RecA mediated hybridization if employed, is an enzymatic strand-pairing reaction, and that conditions normally used for DNA-DNA hybridization (e.g. 0.6 M NaCl) would actually be inhibitory.
  • the precise conditions corresponding to "moderately high stringency" may vary depending on the methodology used to drive the annealing.
  • the term "substantially complementary” includes (1) primers which are perfectly complementary to the target DNA molecule, (2) primers which are complementary for most of their length, but which include one or several mismatches from perfect complementarity, although not enough mismatches to significantly reduce hybridization specificity; and (3) degenerate primers which include several bases at a given site to accommodate a multiplicity of common alleles in the target DNA.
  • the use of mismatched primers may result from the presence of a mutation in the initiation site, or the mismatch may be intentionally selected for introduction of a desired sequence variation into the replicated DNA.
  • the primers used in the invention may also include one or more non- hybridized regions for the purpose of introducing a desired additional sequence into the replicated DNA.
  • this additional sequence may be a sequence which introduces a restriction site near the end of the replicated DNA to facilitate insertion of the replicated copies into other DNA molecules.
  • Preferred restriction sites will be those recognized by rare-cutting restriction enzymes which generally recognize 8-base sequences, or intron- homing endonucleases such as PI- S eel from yeast which recognizes a 31-base pair sequence. This will reduce the likelihood of cleavage occurring within the replicated DNA at other than the intended cleavage site.
  • the primer used comprises a 3 - and a 5' region which are substantially complementary to portions of the target DNA template, and a central non-complementary region which forms a D-loop when the primer is hybridized with the target DNA.
  • a second primer which is complementary is used to form the invading strand of the D-loop. Similar variations for insertion of cleavage sites etc, may be incorporated in the structure of such primers.
  • the primers used in the method of the invention may also include a detectable label or capture moiety.
  • Suitable detectable labels and capture moieties are well known in the art as comparable materials are used in PCR, nucleic acid sequencing, and hybridization-based assays.
  • Specific, non-limiting examples of suitable labels and/or capture moieties include fluorescent dyes such as fluorescein, Texas Red or cyanine dyes; enzyme labels such as alkaline phosphatase; and capturable labels such as biotin. Nucleic acid tails which specifically interact with a known capture sequence can also be employed.
  • the primer is combined with target double- stranded DNA under conditions suitable for hybridization and in the presence of the enzyme RecA, which results in the formation of a D-loop at the site of primer binding.
  • the present invention utilizes replisomes.
  • Replisomes are multi- protein associations which form at a replication fork and act in concert to replicate DNA. Replisomes provide much greater processivity than polymerases used for PCR. For example, the E. coli replisome can synthesize pieces of DNA at least as long as a megabase (1 X 10 6 nucleotides).
  • Replisomes include proteins which perform a variety of functions. Replication of DNA using replisomes depends on an initial unwinding of the DNA duplex at an origin of replication, and the continued unwinding along the strands as the replication process proceeds. This unwinding is carried out by DNA helicases.
  • the resultant regions of single -stranded DNA are stabilized by the binding of single- stranded DNA-binding proteins which are also part of the replisome.
  • the stabilized single- stranded regions are then accessible to the enzymatic activities of polymerases enzymes required for replication to proceed.
  • Replisomes have been shown to be substantially self assembling. Thus, when the necessary proteins are present under appropriate conditions, the replisome will assemble.
  • a preferred combination of proteins for formation of a replisome in accordance with the present invention includes the following proteins:
  • PriA, PriB, PriC, DnaT, DnaB, DnaC primary proteins
  • SSB single- stranded DNA-binding protein
  • DNA polymerase III holoenzyme (Pol III HE).
  • An alternative combination utilizes the mutant protein DnaC810, (described below) in place of PriA, PriB, PriC and DnaT.
  • Pol III HE may be used in a form recovered directly by purification from E. coli, or as a combination of Pol III* and the ⁇ subunit. Pol III HE may also be reconstituted from individually overexpressed and purified subunits. These subunits are ⁇ (DnaE), ⁇ (DnaQ), ⁇ (HolE), ⁇ (DnaN), ⁇ (DnaX, full length), ⁇ (DnaX, truncated), ⁇ (HolA), ⁇ ' (HolB), ⁇ (HolC) and ⁇ (HolD). Preparation of Pol III HE is described in US Patents Nos. 5,668,004 and 5,583,026 which are incorporated herein by reference for those countries in which such incorporation is permitted.
  • Replisomes have been found to initiate DNA replication at the site of a D- loop.
  • the D-loop formed by the interaction of the primer with the target DNA molecule serves as the initiation site for the replication process in accordance with the invention.
  • nucleic acid monomers i.e., deoxynucleotide triphosphates, dATP, dCTP, dGTP and dTTP
  • ATP deoxynucleotide triphosphates
  • target region may be a particular gene, or a particular portion of a gene depending on the use for which the copied
  • DNA is intended.
  • the ability to produce copies of very large numbers of bases changes the practical limits on the proximity between the primer and the target region from those which are usually observed in the PCR and comparable methods.
  • the initiation site must be "adjacent" to the target region, this means only that the initiation site must be close enough to and on the correct side of the target region such that a replisome assembled at the
  • D-loop will copy the DNA of the target region.
  • the first primer is as described above, and hybridizes with a first strand of a double stranded DNA duplex.
  • the second primer also is a substantially complementary oligonucleotide primer, but it hybridizes to the second strand of the DNA duplex at a second initiation site located on the other side of the target region.
  • the two primers flank the target region, in the same manner that PCR primers flank a region to be amplified.
  • the same principle which leads to amplification of just the region bounded by PCR primers leads to creation of much larger pieces of replicated DNA spanning the region between the two initiation sites using the method of the invention, although the efficiency may not be as great as achieved with PCR.
  • DNA replicated in accordance with the invention may be utilized for a variety of purposes.
  • the replicated DNA may be used as a source of genetic material to be spliced into still larger nucleic acid constructs, including plasmids, cosmids, viral vectors etc., to facilitate expression of the replicated DNA in a suitable host system
  • Such splicing can be facilitated by the incorporation of restriction sites near then ends of the replicated DNA as discussed above.
  • restriction sites can be introduced at both ends of the replicated DNA.
  • the replication of DNA in accordance with this method can be used as part of a method for detecting genomic rearrangements in a target DNA sequence.
  • a D-loop is introduced into the DNA at a selected initiation point, a replisome is assembled at the D-loop, and the DNA is copied to produce sufficient numbers of copies for analysis.
  • the copied product is analyzed to detect variations in size or organization of the copied material using size-specific separations, hybridization probes and other standard analytical techniques. It will be appreciated that the use of size-specific separations requires the production of a product of defined lengths, and thus will generally require the use of the two primer embodiment discussed above.
  • the analysis involves the measurement of the interaction of the DNA with a labeled or immobilized probe, the replication of multiple copies of a single strand of the DNA, without amplification, may be sufficient.
  • the method can be used to facilitate linkage mapping.
  • the method can be used in the circumstance where two chromosomal markers are known to be near one another, but where the exact distance separating them is not known.
  • D-loop oligonucleotide primers are synthesized for each marker for both the DNA strands.
  • Combinations of the primers are used to replicate the region between the two markers, and the size of the product formed reflects the chromosomal distance between the two markers.
  • the method may also be used to map unlinked genes, and markers such as RFLPs. SNIPs and ESTs.
  • a dnaC810 open reading frame was constructed by splicing overlap extension polymerase chain reaction and cloned into the Ndel site of the pETl 1C overexpression plasmid (Novagen). Overexpression and purification of DnaC810 was as for the wild type protein.
  • PriA, PriB, PriC, DnaT, DnaB and DnaC were purified by the methods described in Marians, K.J. Methods Enz mol. 262: 507-521 (1995).
  • SSB was purified using the procedures described in Minden and Marians, J. Biol. Chem. 260: 9316-9325 (1985).
  • the DNA polymerase III holoenzyme was either reconstituted from Pol III* and ⁇ subunit as described by Wu et al. /. Biol. Chem. 267: 4030-4044 (1992) or from purified subunits as described in Marians et al., J. Biol. Chem. 273: 2452-2457 (1998).
  • EXAMPLE 2 To validate the operability of the inventive concept, a double-stranded circular template DNA was prepared in accordance with the steps shown in Fig. 1. A 100 nt-long oligonucleotide primer (Seq. ID No. 1) was annealed to flR408 viral DNA (Russell et al., Gene 45: 333-339 (1986)). The central 42 nt of this oligonucleotide are non-homologous with the template, thus forming a D-loop in the resulting heteroduplex.
  • MgOAc 10 mM DTT, 80 mM KC1, 200 ⁇ g/ml bovine serum albumin, 2 mM ATP, 40 ⁇ M dNTPs, 0.42 nM [ 32 P] form II D loop DNA template, 0.5 ⁇ M SSB, 225 nM DnaC, 30 nM DNA polymerase III holoenzyme, PriA, PriB, PriC, DnaT and DnaB were incubated at 37°C for 10 minutes.
  • reaction mixture was also performed in which one of the proteins (PriA, PriB, PriC, DnaT, DnaC and DnaB) was omitted in each reaction mixture.
  • template alone and template with the holoenzyme alone were also evaluated.
  • Reactions were terminated by the addition of EDTA to a concentration of 25 mM and NaOH to a concentration of 50 mM.
  • the reaction products were evaluated by electrophoresis at 2 V/cm for 20 hours at room temperature through horizontal 0.7% alkaline agarose gels using 30 mM NaOH, 2 mM EDTA as the electrophoresis buffer. The gels were neutralized, dried and analyzed by autoradiography.
  • the electrophoresis gels showed that incubation of the D-loop template, the seven primosomal proteins, SSB and DNA polymerase III holoenzyme resulted in extension of the invading strand oligonucleotide (42 nt, Seq. ID. No. 2) to the full length template size
  • extension of the invading strand could result from one of two processes: either (1) assembly of a bonafide replication fork at the D loop followed by elongation of the leading strand coupled with unwinding of the duplex DNA template, or (2) uncoupled unwinding of the template DNA leaving an oligonucleotide annealed to the viral single stranded DNA that could be elongated in a primer extension reaction by the polymerase.
  • coupled replication fork action requires a protein- protein interaction between DnaB and the ⁇ subunit of the holoenzyme. Kim et al.., Cell 84: 643-650 (1996). In the presence of this interaction, replication forks could move rapidly, at nearly 1000 nt/sec, whereas in its absence, the polymerase becomes stuck behind a slow- moving helicase and replication fork progression proceeds at only about 30 nt/sec.
  • K230A and K230D substitutions were tested. All three supported replication on the D loop to a greater extent than the wild-type protein. This same type of improved activity in the mutant proteins has been observed in other systems (Zavitz, supra), and may arise because the mutant proteins remain bound to the site of DNA binding, providing a better target than the wild- type protein that can move off the site because of its helicase activity.
  • EXAMPLE 6 E. coli strains carrying priA mutations are very difficult to grow. They are rich-media sensitive, form huge filaments, and have a viability roughly one-hundredth that of the wild-type. Sandier et al., Genetics 143: 5-13 (1996); Nurse et al, J. Bacteriol. 6686-
  • DnaC and DnaB did not support elongation of the invading strand of the D loop.
  • DnaC810 was clearly able to load DnaB to the D loop on the template in the absence of the other primosomal proteins, as evidenced by the elongation of the invading strand to full length.
  • the E176G substitution in DnaC810 represents a true gain of function mutation that allows bypass of the DnaB loading pathway that involves PriA, PriB, PriC and DnaT and permits a reduction in the number of proteins necessary for the practice of the present invention.
  • EXAMPLE 7 Construction of Plasmid pETl lc-dnaC810—A dnaC810 open reading frame (ORF) was made by two-step overlapping polymerase chain reaction (PCR) Morton et al, Gene 77: 61-68 (1989). The N- terminal coding region of dnaC810 was PCR amplified using plasmid pETl Ic-dnaC (Marians, K.J, Methods Enzymol. 262:m 507-521 (1995)) as a template and two flanking primers:
  • Ndel primer (Seq. ID No. 3), which carries a Nd l site at the dnaC initiator codon, and ( ⁇ ) the Agel' primer (Seq. ID. No. 4), which carries the designed point mutation (E176G,
  • the C- terminal coding region of dnaC810 was also PCR amplified using plasmid pETl Ic-dnaC as a template and two different flanking primers:
  • the gel purified dnaC810 ORF fragment was digested with Ndel and BamHI and ligated with Ndel- and R ⁇ mHI-digested pETl lc plasmid DNA to give pETl lc-dnaC810.
  • This suspension was centrifuged at 100,000 x g for 1 h (Sorvall T865 rotor).
  • the supernatant fraction 1, 65 ml, 3510 mg protein
  • the supernatant was adjusted to 0.04% polymin P by dropwise addition of a 1% solution.
  • the precipitate was removed by centrifugation at 47,000 x g in a Sorvall SS-34 rotor for 30 min.
  • the supernatant was further subjected to (NH SO-ifractionation (50% saturation) by the addition of solid.
  • the resulting protein pellet was collected by centrifugation at 47,000 x g in a Sorvall SS-34 rotor for 30 min.
  • the protein pellet was resuspended in 8 ml of buffer A [50 mM Tris-HCl (pH 7.5 at 4 °C), 1 mM EDTA, 5 mM dithiothreitol, 20% glycerol, 0.01% Brij 58] + 50 mM NaCl to give fraction 2 (13 ml, 1108 mg protein).
  • Fraction 2 was dialyzed against 2 1 of buffer A + 50 mM NaCl for 12 h and then loaded onto a 100-ml DEAE- cellulose column (4 cm x 20 cm) that had been equilibrated previously with buffer A + 50 mM NaCl. The column was washed with 200 ml of buffer A + 50 mM NaCl.
  • Fractions (15 ml) of the flow-through and wash that contained protein were pooled to give fraction 3 (81 ml, 363 mg protein).
  • Fraction 3 was loaded directly onto a 35-ml SP-Sepharose FF column (formed in a 60-ml disposable syringe) that had been equilibrated previously with buffer A +
  • Fractions (1 ml) containing DnaC810 were pooled to give fraction 6 (7.5 ml, 9.2 mg protein).
  • Fraction 6 was then loaded onto a 3-ml phosphocellulose column that had been equilibrated with buffer A + 50 mM NaCl. The column was washed with 6 ml of equilibration buffer and protein was eluted with a 60-ml linear gradient of 50-400 mM NaCl in buffer A.
  • DnaC810 eluted at 250 mM NaCl (Fraction 7, 3.5 ml, 5.2 mg protein).
  • ATGAGCTCCA TATGCTAGCT AGGGAGGCCC CCGTCACAAT CAATAGAAAA TTCATATGGT TTACCAGCGC

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Abstract

Cette invention concerne une méthode de réplication d'ADN, notamment la réplication de grands brins d'ADN. On combine une amorce avec une molécule cible d'ADN à répliquer. L'amorce est conçue de façon à être au moins en partie homologue d'un site connu de l'ADN cible et de façon à créer une boucle D lorsqu'elle est hybridée avec ce site. On assemble ensuite un réplisome à la boucle D et ce réplisome produit une copie de l'ADN en commençant au site de liaison de l'amorce. On peut répliquer de grands brins d'ADN de façon comparable à l'amplification par polymérase lorsque l'on utilise deux espèces d'amorces de boucle D qui se lient à des sites éloignés sur l'ADN et qui flanquent une région devant être répliquée. On peut analyser l'ADN répliqué pour détecter des variations dans la séquence génétique de la cible, pour établir des cartes de liaison ou pour fournir une source de molécules d'ADN plus longues présentant une séquence souhaitée.
PCT/US2000/004445 1999-02-04 2000-02-03 Procede de replication d'adn Ceased WO2000046408A1 (fr)

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