WO2007056173A1 - Amplification de génome - Google Patents

Amplification de génome Download PDF

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Publication number
WO2007056173A1
WO2007056173A1 PCT/US2006/043014 US2006043014W WO2007056173A1 WO 2007056173 A1 WO2007056173 A1 WO 2007056173A1 US 2006043014 W US2006043014 W US 2006043014W WO 2007056173 A1 WO2007056173 A1 WO 2007056173A1
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dna
polymerase
amplification
duplex
fragments
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PCT/US2006/043014
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English (en)
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Rebecca Kucera
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New England Biolabs, Inc.
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Publication of WO2007056173A1 publication Critical patent/WO2007056173A1/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
    • C12Q1/6846—Common amplification features

Definitions

  • a method for rapid amplification of a large duplex DNA that includes (a) adding to the large duplex DNA in the absence of primers: (i) one or more nicking endonucleases for creating a plurality of nicks in both strands of the duplex DNA without substantially fragmenting the DNA into discrete small fragments; and (ii) one or more DNA polymerases; (b) allowing the one or more DNA polymerase to initiate DNA synthesis from the 3 1 OH termini of the large duplex DNA; and (c) obtaining gel electrophoresis detectable amounts of amplified DNA within a reaction time of less than about 10 minutes for example 5 minutes or less, using for example, 25ng of starting material in a 50 ⁇ l reaction volume.
  • Embodiments of the method permit hybridization between single-stranded DNA to form the large duplex DNA and can provide substantially proportional representation of sequences in the large duplex DNA.
  • the large duplex DNA may be all or part of a genome such as a phage, bacterial or mammalian genome.
  • the one or more nicking endonucleases have a recognition sequence specificity for four or more nucleotides where examples of nicking enzymes include N.BstNBI, N.BbvCIA; N.BbvCIB, NB.MIyl, NB.SapI, and Nb.Bsml nicking endonucleases.
  • the one or more DNA polymerases may include a Family A polymerase for example, Bst polymerase large fragment, T7 polymerase and E. coli polymerase large fragment, or a Family B polymerase for example, phi 29 polymerase, or a hyperthermophilic archaeal polymerase.
  • the amplification product includes any or all of a single-stranded DNA fragment released from the large duplex DNA, double-stranded DNA fragments or DNA fragments that are partially single-stranded and partially double- stranded.
  • the amplification product may further include a plurality of sets of fragments wherein each set of fragments contains a plurality of fragments with overlapping sequences.
  • the method includes an additional step of adding primers to anneal to denatured duplex DNA and/or single-stranded DNA to increase the concentration of selected sequences in the duplex DNA.
  • a method is provided for obtaining a fingerprint of a large duplex DNA where the large duplex DIMA may be part or all of a genome. The method includes the steps of amplifying large duplex DNA using methods described above where the nicking endonucleases are pre-selected and determining the quantity and size of different DNA fragments produced during amplification to provide the fingerprint for the duplex DNA.
  • the preselection of nicking endoucleases may be achieved by analyzing the sequence of the duplex DNA and determining the frequency of nick sites on each strand of the duplex.
  • the quantity and size of the fragments produced during amplification can be determined by gel electrophoresis. This method may be used in a diagnostic test to determine the identity of DNA in a biological or environmental sample.
  • different related DNAs can be characterized by fingerprints, which can be compared. Differences in the fingerprints can be correlated with the presence of a mutation where the mutation can be a sequence variation, a deletion or an insertion.
  • a method for detecting a target sequence in a duplex DNA that includes (a) obtaining an amplification product according to any of the above described methods; (b) selecting primers for primer-based amplification of the gene sequence; and (c) amplifying the particular gene sequence using the primers in (b) for detecting the target sequence.
  • This method may be used in a diagnostic test to determine the identity of DNA in a biological or environmental sample.
  • a DNA preparation is provided that includes multiple copies of DNA fragments that are representative of a substantially whole genome. These DNA fragments preferably provide a consistent pattern of bands on an ethidium bromide agarose gel, the pattern of bands representing a fingerprint of the substantially whole genome.
  • Figure 1 shows the mechanism of nick-based large duplex DNA amplification.
  • Step 1 Nicking enzyme cleaves DNA in each strand in or close to its recognition site (*), generating an upstream 3'-OH available for primer extension by a DNA polymerase and a downstream 5'-PO 4 .
  • Step 2 DNA polymerase initiates synthesis by binding to the 3'-OH terminus at the nicked site. (Action shown on one strand for clarity, but any nicked site on either strand could support initiation of DNA synthesis.)
  • Step 3 Synthesis is initiated, strand-displacing the encountered downstream DNA.
  • Step 4 Synthesis and strand-displacement continues until the DNA polymerase encounters a nick in the template strand.
  • the original recognition site for the nicking enzyme has been regenerated during this process, and the original displaced strand remains tethered to the DNA substrate.
  • Step 5 Subsequent nicking, followed by DNA polymerase synthesis and strand-displacement of encountered DNA 7 frees the previously synthesized single-stranded DNA.
  • Step 6 This entire process as described in steps 1-5 also occurs on the opposite strand.
  • Step 7 Most of the amplification product is single-stranded in nature. However, if two nicks occur at symmetrical or near symmetrical locations on opposite strands, as shown in this figure, the synthesized and displaced strands can anneal to form double-stranded products or products substantially double-stranded in nature.
  • Figure 2 shows a diagram illustrating the nicking sites and some examples of possible DNA synthesis products in lambda phage DNA.
  • Lightly shaded arrows indicate some of the single-stranded products of large duplex DNA that arise from synthesis at a nick indicated by vertical lines (
  • Figure 2 shows multiple initiation sites in an overlapping family of fragments.
  • Figure 2(1) and (9) are single-stranded fragments that are amplification products at the end of a large DNA. Depending on the distribution of nick sites, it might be expected that more generally, amplification products will be single-stranded at the ends of a duplex DNA.
  • Figure 2(2), (4), (5), (6), (7) and (8) show pairs of different sized amplification products corresponding to amplification between two nick sites on complementary strands. These products are double-stranded.
  • Figure 2(3) shows a nested family of fragments that reflect different start sites on one strand of the duplex that result from the status of the nick site.
  • Various sized amplification products can also be produced on the complementary strand of the duplex generating double-stranded DNA products and products that are partially double-stranded and partially single-stranded.
  • Double-stranded DNA corresponding to Figure 2(2)-(8) form the major bands observed on an agarose gel.
  • Figure 3 shows a time course of amplification products obtained with a large duplex DNA exemplified by lambda phage DNA. Major products are indicated by astericks.
  • Lane 1 NEB 2-log ladder DNA marker (NEB product #N3200L, New England Biolabs, Inc., Ipswich, MA); Lane 2: nWGA product synthesis, 0 min, 50 ng/50 ⁇ l vol;
  • Lane 3 nWGA product synthesis, 10 min, 50 ng/50 ⁇ l vol;
  • Lane 4 nWGA product synthesis, 20 min, 50 ng/50 ⁇ l vol;
  • Lane 5 nWGA product synthesis, 30 min, 50 ng/50 ⁇ l vol; Lane 6: NEB 2 log ladder DNA marker (New England Biolabs,
  • Lane 7 nWGA product synthesis, 0 min, 25 ng/50 ⁇ l vol;
  • Lane 8 nWGA product synthesis, 5 min, 25 ng/50 ⁇ l vol;
  • Lane 9 nWGA product synthesis, 10 min, 25 ng/50 ⁇ l vol; Lane 10: nWGA product synthesis, 20 min, 25 ng/50 ⁇ l vol;
  • Lane 11 nWGA product synthesis, 30 min, 25 ng/50 ⁇ l vol.
  • Figure 4 shows a sensitivity determination of the amount of input DNA required to generate identifiable product in 30 and 75 minutes at 45°C.
  • Lane 1 0 ng input lambda DNA, 30 min at 50 0 C;
  • Lane 2 1 ng input lambda DNA, 30 min at 50 0 C;
  • Lane 3 10 ng input lambda DNA, 30 min at 50 0 C;
  • Lane 4 25 ng input lambda DNA, 30 min at 50 0 C; Lane 5: 50 ng input lambda DNA, 30 min at 50 0 C;
  • Lane 8 0 ng input lambda DNA, 75 min at 45°C;
  • Lane 9 1 ng input lambda DNA, 75 min at 45°C;
  • Lane 10 5 ng input lambda DNA, 75 min at 45°C;
  • Lane 11 10 ng input lambda DNA, 75 min at 45°C.
  • Figure 5 shows that different nicking enzymes can be used for nWGA.
  • Lane 1 NEB 2-log ladder DNA marker (New England Biolabs,
  • Lane 2 Negative control (identical to lane 3 below, but no incubation period at 50 0 C);
  • Lane 5 N.BbvCIB
  • Lane 6 N.BbvCIB variant 1-37
  • Lane 7 NB.MIyl
  • Lane 8 NB.SapI
  • Lane 11 Nb.Bsml, 10 units, 65°C assay temperature
  • Figure 6 shows that different DNA polymerases can be used for rapid large DNA amplification.
  • Lane 1 NEB 2-log ladder DNA marker (New England Biolabs,
  • Lane 2 9 0 N (modified) DNA polymerase, assay temperature of 45°C
  • Lane 3 9°N (modified) DNA polymerase, assay temperature of 5O 0 C;
  • Lane 4 9 0 N (modified) DNA polymerase, assay temperature of 55°C;
  • Lane 5 9°N (modified) DNA polymerase, assay temperature of 50 0 C, T4gp32 from a different source
  • Lane 6 9 0 N (modified) DNA polymerase, assay temperature of 45°C, + additional 25 mM KcI;
  • Lane 7 9 0 N (modified) DNA polymerase, assay temperature of 45°C, with a preincubation step for the polymerase and N.
  • Lane 8 Bst DNA polymerase, assay temperature of 50 0 C;
  • Lane 9 Bst DNA polymerase, assay temperature of 50 0 C, with a preincubation step as described in Lane 7 above.
  • Figure 7 shows the effectiveness of rapid large DNA amplification with different sized genome samples other than the previously described lambda DNA phage genome.
  • Lane 1 T7 viral DNA (10,674 bp);
  • Lane 2 a laboratory plasmid construct (pUC19 containing the
  • Lane 3 E. coli genomic DNA
  • Lane 4 NEB 2-log ladder DNA marker (New England Biolabs,
  • Figure 8 shows potential uses for rapid large DNA amplification.
  • Figure 8A Viral genome is shown here as a template for rapid large DNA amplification (1) and gives rise to a fingerprint pattern against a low level smear (2) unique to that DNA.
  • Figure 8B Bacterial or eukaryotic genome can serve as a template for rapid large DNA (3) and gives rise to a high molecular weight smear with faint double-stranded DNA fragments visible (4). Such material can then be used for target-specific amplification (5a) or detection (5b).
  • Figure 9 shows the fingerprints obtained with large genome samples, such as various E. coli strains, when used as template DNA for rapid large DNA amplification.
  • Lane 1 NEB 2-log marker DNA ladder (New England Biolabs, Inc., Ipswich, MA);
  • Lanes 2, 3 ERl 506 E. coli strain; Lanes 4, 5: ER1793 E. coli strain; Lanes 6, 7 : ER2592 E. coli strain; Lanes 8, 9: ER2609 E. coli strain; Lanes 10, 11 : ER2612 E. coli strain;
  • Lanes 12, 13 ER2613 E. coli strain
  • Lanes 14, 15 ER2714 E. coli strain
  • Lanes 16, 17 NEB 2-log marker DNA ladder (New England Biolabs, Inc., Ipswich, MA); Lanes 18, 19: ER2729 E. coli strain;
  • Lane 20 NEB 2-log marker DNA ladder (New England Biolabs, Inc., Ipswich, MA).
  • Figure 10 shows that downstream diagnostic detection by target-specific PCR amplification can be performed on DNA generated by nWGA.
  • Lane 1 1 ⁇ g lambda-Hind III digested marker DNA (NEB product #N3012L (New England Biolabs, Inc., Ipswich, MA); Lane 2: E. coli cell strain ER1505, unamplified DNA, 20 cycles;
  • Lane 3 same as lane 2, except 25 cycles;
  • Lane 4 E. coli cell strain ER1505, nWGA DNA, 20 cycles;
  • Lane 5 same as lane 4, except 25 cycles;
  • Lane 6 E. coli cell strain ER2612, unamplified DNA, 20 cycles; Lane 7: same as lane 6, except 25 cycles;
  • Lane 8 E. coli strain ER2612, nWGA DNA, 20 cycles;
  • Lane 9 same as lane 8, except 25 cycles;
  • Lane 11 E coli cell strain ER1505, nWGA DNA, 25 cycles, 1.25 units Taq DNA polymerase used for the PCR;
  • Lane 12 same as lane 11 except 2.5 units Taq DNA; polymerase used for the PCR;
  • Lane 13 same as lane 11; Lane 14: same as lane 12;
  • Lane 15 E. coli cell strain ER2612, nWGA DNA, 25 cycles,
  • Lane 16 same as lane 15 except 2.5 units Taq DNA polymerase used for the PCR; Lane 17: same as lane 15;
  • Lane 18 same as lane 16;
  • Figure 11 shows the reproducibility of the mWGA process. Individual 50 ⁇ l reactions containing 25 ng lambda DNA were incubated at 5O 0 C for 30 minutes.
  • Lane 6 nWGA product synthesis
  • reaction 6 Lane 7: NEB 2-log ladder DNA marker (NEB product #N3200L,
  • DNA product synthesized from a large DNA template may be predominantly single-stranded.
  • the amplification product however may be a mixture of single-stranded and double-stranded DNA or may be substantially double-stranded.
  • Double-stranded DNA may be formed as a result of symmetrical or near symmetrical positioning of nicking sites on the two strands of the double-stranded DNA. Because of the reproducibility of this type of amplification, it can be used for fingerprinting DNA such as whole genomes. It can also be used for diagnostic purposes for detecting changes in large DNAs caused for example by mutation. It can also be used for generating large amounts of DNA for further downstream applications.
  • “Large duplex DNA” refers to DNA that is at least approximately lkb but may be 5kb or larger, 10kb or larger, 20kb or larger, 30kb or larger, 40kb or larger, 50kb or larger, 60kb or larger, 70kb or larger, 80kb or larger, or 90kb or larger.
  • Large duplex DNA is intended to include part or all of vectors and plasmids, or genomes from viruses, prokaryotics or eukaryotics.
  • “Duplex DNA” is predominately double-stranded but may also include some single- stranded regions.
  • Fingerprint refers to a unique characteristic of a DNA sample. A reproducible pattern of fragments from a DNA can be obtained on a gel from which it is possible to determine the identity of large DNA.
  • Status of a nick site refers to whether the nick site on a DNA is in a nicked or non-nicked state, a condition which is dynamic.
  • any one nicking site in the DNA template is constantly changing its status from being intact and available for binding and nicking by a nicking enzyme to being nicked by the nicking enzyme, which has become dissociated from the site, permitting the binding and extension of the free 3'-OH end by a DNA polymerase in the reaction mix.
  • This extension by the polymerase regenerates the nicking enzyme recognition site, reverting the status of the nicked site back to its original intact state, once again available for binding and nicking by the nicking enzyme.
  • Th e status of any one nick site has a direct effect on polymerase involvement at that nick, and also an indirect effect as a termination site with regard to any primer extension from a nick on the opposite strand (the template strand) downstream from the first nick.
  • Primer extension from any nicked site is terminated, and the single-stranded DNA displaced, when a nick is encountered in the template strand.
  • the status of the nick site in the template strand whether intact, nicked, or intact due to regeneration, allows a full family of related single-stranded DNA sequences to be produced, differing only when a nicked site is encountered on the template strand (see Figure 2).
  • the amplification reaction can also be optimized for a particular sequence of interest (target) by choosing an appropriate nicking enzyme(s) that does not nick in the target DNA sequence.
  • a nicking endonuclease for use in amplification including whole genome amplification may be selected according to one or more of the following considerations:
  • nicking DNA at more than a few sites but not at too many sites would decrease the amplification efficiency and would promote a high level of sequence drop-outs; too many cut sites would generate a preponderance of very small DNA fragments, obviating the accumulation of longer sequences that may be desired for the downstream use of the DNA.
  • a nicking endonuclease may preferably nick at more than about 30 sites but less than about 200 sites along the duplex DNA.
  • lambda genome is nicked 35 times on the top strand and 26 times on the bottom strand (total of 61 times) or approximately 1-2 sites/1000 bases to give rise to whole genome amplification;
  • Examples of nicking endonucleases for use in the whole genome amplification method may include any one or more of the following: N.BstNBI (NEB Catalog #R0607, New England Biolabs, Inc., Ipswich, MA), Nt/Nb BbvCI (New England Biolabs, Inc., Ipswich, MA), Nt.AlwI (NEB Catalog #R0627, New England Biolabs, Inc., Ipswich, MA), Nb/Nt Bsal (U.S. Publication No. 2005-0136462), Nb.Bsml (International Publication No. WO 2006/012593), Nb/Nt Sapl (U.S. Publication No.
  • Nb/Nt BpulOl Fermentas Inc., Hanover, MD
  • Nval269 Fermentas Inc., Hanover, MD
  • Nt.Mlyl U.S. Patent No. 6,395,523
  • Nt.BhalllP Nb.BpulOI, Nbt.BpulOIB, Nb/Nt BsmAI, Nt.BsmBI, Nt.BspCAI, Nt.BspD61, Nb.BsrDI, Nt.Bst91, Nb.BstNBIP, Nt.EsaSS1198P and Nt.BstSEl (Rebase - http://rjr.neb.com/cqi-bin/azlist?nick ' ).
  • polymerases for use in the nick-based amplification method include one or more of a Family A polymerase or a Family B polymerase that are preferably capable of strand- displacement as well as initiation at a nick site.
  • polymerases for use in the amplification method include phi29 DNA polymerase, Bst DNA Polymerase, Vent ® DNA polymerase, Vent ® (exo-) DNA polymerase, Deep VentTM polymerase, 9 0 N polymerase and modifications thereof, E. coli Poll (Klenow fragment), Klenow fragment 3'-5' exo-, M-MuLV Reverse Transcriptase and Thermomicrobium roseum DNA pol I large fragment. It may be desirable to use a mixture of more than one polymerase to optimize both initiation of polymerization at a nick site on the DNA and strand- displacement activities at a desired temperature.
  • the ratio of nicking endonuclease to polymerase for obtaining optimum amplification for a particular DNA can be determined by varying the concentrations of each enzyme relative to the other and analyzing the results by gel electrophoresis. Once established, the enzyme ratio can be used for all amplification reactions involving the particular DNA.
  • Embodiments of the method can be used for diagnostic purposes and more specifically for fingerprinting.
  • Figure 9 shows how different strains of E. coli can be differentiated from each other.
  • a fingerprinting profile made up of a specific number and size of fragments obtained by amplfication and observed by gel electrophoresis, depends on which nicking endonuclease are selected.
  • Fingerprinting profiles can be compared to each other provided that a nicking endonuclease with the same specificity is used for all the samples in the comparison.
  • Figure 10 shows a diagnostic application in which a secondary amplification of a particular gene of interest is carried out after nWGA.
  • Figure 1 illustrates the amplification process.
  • a duplex DNA sample was amplified by supplementing a buffered reaction containing the DNA template of interest with a binding protein, dNTPs, a divalent cation, a DNA polymerase capable of polymerization and strand-displacemen or DNA polymerase mix and an appropriate nicking enzyme or nicking enzyme mix.
  • a buffer consisting of 10 mM KCI, 20 mM Tris-HCI (pH 8.8 at 25°C), 10 mM (NH 4 ) 2 SO 4 , and 0.1% Triton X-100 (ThermoPol II reaction buffer, NEB product #B9005S, New England Biolabs, Inc., Ipswich, MA) was utilized.
  • the buffer was supplemented with 400 ⁇ M each dNTP, 10-20 ⁇ g of T4gp32 binding protein (NEB product #M0300L, New England Biolabs, Inc., Ipswich, MA), 5-6 mM MgCb, 10 units of Bst DNA polymerase (NEB product #M0275L, New England Biolabs, Inc., Ipswich, MA), 4-8 units of N.BstNBI nicking enzyme (NEB product #R0607L, New England Biolabs, Inc., Ipswich, MA) and 1-50 ng lambda DNA (NEB product #N3011L, New England Biolabs, Inc., Ipswich, MA) per 50 ⁇ l reaction volume, and placed at 45-50°C for 20-75 minutes.
  • the reaction was stopped by the addition of a buffered SDS/EDTA solution. Specifically, 1/10 volume of the reaction was added consisting of 25% glycerol, 100 mM tris (pH8), 100 mM EDTA (pH 8), 0.2% SDS and 0.15% bromophenol blue tracking dye.
  • reaction mix 25 ⁇ l was electrophoresed with a 1.2% agarose gel run at 80 milliamps in standard tris-borate-EDTA (TBE) buffer, or was available for further downstream diagnostic uses.
  • TBE tris-borate-EDTA
  • Example 2 Large DNA amplification of a 48.5kb genome
  • Example 3 Assay to determine the sensitivity of rapid large PNA amplication to the starting concentration of PNA
  • different amounts of lambda DNA were added to ThermoPol II buffer as described in Example 1, supplemented with 40OuM dNTPs and 10 units Bst DNA polymerase. Additional components were as follows: for lanes 1-5, 10 ⁇ g Tg4p32 binding protein, 5 mM MgCI 2 , and 8 units of N.BstNBI nicking enzyme; for lanes 8-11, 20 ⁇ g T4gp32 BP, 6 mM MgCI 2 and 4 units of N.BstNBI nicking enzyme. The mixtures were incubated at 45-50 0 C for 30-75 minutes. To end the assay, 20 ⁇ l samples were added to a buffered SDS/EDTA solution and electrophoresed as described in Example 1.
  • nicking enzymes can support rapid large DNA amplification.
  • any particular DNA substrate may possess recognition sites for a variety of nicking enzymes. The frequency and position of these sites on each strand result in a different banding pattern due to primary double-stranded products and a different overall amplification factor.
  • a variety of nicking enzymes were tested with the lambda bacteriophage DNA substrate as described in Example 1. 6 mM MgCb was used unless otherwise specified. The incubation time was 5 minutes unless otherwise specified, and the temperature was 50 0 C unless otherwise specified. At the end of the incubation period, the samples were processed as described in Example 1. The results are shown in Figure 5. While this example shows the use of single nicking endonucleases, the use of multiple nicking endonucleases may be desirable to obtain an appropriate distribution of nicking sites where the characteristics of an appropriate number of nick sites are discussed in the detailed description.
  • Two different DNA polymerases are here shown to support rapid large DNA amplification.
  • any particular DNA polymerase that can initiate polymerization at a nick and strand- displace encountered DNA at the same temperature as a nicking enzyme can be used in the amplification method.
  • two thermophilic DNA polymerases were tested for the ability to function in nWGA.
  • the assay temperature unless otherwise indicated was 45°C and the assay incubation time was 2 hours. Other variables were examined in this early experiment and are labeled as such. These included the assay temperature, testing another source of T4gp32 binding protein, the addition of KCI or the effect of a possible pre-incubation step of the enzymes used in the amplification method with frequent cutting restriction enzymes to decrease the background amplification due to contaminating DNA in the enzyme reagents. These preincubations were performed in 20 ⁇ l of the final 30 ⁇ l reaction volumes, in ThermoPol buffer for 5 minutes at 30 0 C before having the restriction enzymes heat inactivated by exposure to 65°C for 5 minutes (see Figure 6).
  • Figure 6 shows that even under non-optimized conditions, 9°N DNA polymerase in the presence of buffer containing an extra 25 mM of KCI and Bst polymerase effectively supported nWGA (see Lanes 6 and 8).
  • the rapid large DNA amplification process has been used with a variety of different sizes of DNA genomes.
  • 50 ⁇ l reaction volumes containing ThermoPol II buffer as described in Example 1 400 ⁇ M dNTPs, 10 ⁇ g Tg4p32 binding protein, 6 mM MgCI 2 , 100 ⁇ g/ml BSA, 10 units Bst DNA polymerase, 4 units of N.BstNBI nicking enzyme, and 25 ng of a variety of DNA substrates were added.
  • the reactions were incubated at 45 0 C for 30 minutes, and processed as described in Example 1.
  • the results are shown in Figure 7. With complex genome templates such as the E. coli genomic DNA in Lane 3, most of the DNA synthesized is in the form of single-stranded DNA.
  • any nick site may be nicked, or have undergone synthesis by the DNA polymerase, which regenerates the un-nicked site. Since the synthesis of any one strand will be halted upon reaching a nicked site in the template strand, different lengths of synthesized product will be made from synthesis originating from a nick site as the DNA polymerase reads the template strand and reaches a site that may or may not be nicked at that moment.
  • Example 7 Diagnostic uses of rapid large DNA amplification
  • Figure 8 illustrates how small genomes, such as viral genomes, for example, lambda bacteriophage or T7 or cloning plasmids such as pUC19 constructs containing different sized inserts (also described in Example 6) can be amplified to give primary product double-stranded DNA fragments against a background of single-stranded products unique for that DNA genome (Figure 8(2)).
  • This profile of the primary product can be a signature that characterizes the identity of the DNA.
  • the signature is a diagnostic tool suitable among other uses for fingerprinting.
  • the amplification process can also be used for complex genomes such as bacterial or eukaryotic genomes.
  • amplification yields a predominance of single-stranded products although some double-stranded DNA fragments can be seen against the single-stranded background ( Figure 8(4)).
  • Amplification results in a type of identifying fingerprint that is sufficient to differentiate E. coli bacterial strains as shown in Figure 9.
  • the amplified material without electrophoresis, can also be used in secondary amplification, which is target- specific and uses designated primers in the PCR process Figure 8(5)) or any type of DNA sequence detection assay Figure 8(6)).
  • the amplified E. coli DNA was generated as described in Example I except MgCb was 6 mM and E. coli DNA from two different cell strains was present at 25 ng (unless otherwise noted) per 50 ⁇ l reaction volume for 45°C for 60 minutes.
  • the amplified material was subjected to QiaQuick (Qiagen Inc., Valencia, CA) purification and quantitated by determining the OD at 260 nM on a Beckman DU-600.
  • Both unamplified and amplified DNA were used as template in a PCR reaction consisting of ThermoPol buffer, 200 ⁇ M dNTPs, 1.25 units of Taq DNA polymerase (NEB product #M0267L, New England Biolabs, Inc., Ipswich, MA) and 0.4 ⁇ M of each forward and reverse primer as described below:
  • AIk B gene (1242 bp PCR product; no N.BstNBI nicking site within the gene)
  • fw primer 5'-CGGTAAACGAAGTGATGCCC-3' (SEQ ID NO: 1)
  • rv primer 5'-CATTCGCGGCACTGC I I I I C-3' (SEQ ID NO: 2)
  • OmpT gene (1797 bp PCR product; one nick site within the gene)
  • fw primer 5- I GTGGCTATAACAGTACTTC-3 I (SEQ ID NO:3)
  • rv primer 5 I -GCGGCCCACGACTTAGAAG-3 I
  • rv primer 5'-AAGAGTATTCCAGCCTGACG-S' (SEQ ID NO:6)
  • the PCR protocol consisted of 94°C for 2 minutes as a pre- soak; then 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes (AIkB and Pgm gene products) or 2 minutes (OmpT gene products), followed by a 4 minutes soak at 72 0 C. 20 ⁇ l from the 50 ⁇ l PCR reaction volumes was removed for gel analysis as described in Figure 1 at both 20 and 25 cycles. The results are shown in Figure 10.
  • Lanes 2-9 of Figure 10 show that the yield of amplicons from the original unamplified DNA and the amplified DNA from two different E. coli cell strains are within a factor of two, indicating a strong level of representation between the original (unamplified) and the rapid large DNA amplification material, when the amplicon did not contain a recognition site for the enzyme used for nicking in rapid large DNA amplification.
  • large amplicons were generated by target-specific amplification, specific downstream diagnostic information was obtained on DNA generated by rapid large DNA amplification, even if a nicking site exists within the target gene sequence.
  • the specific amplicon is produced regardless of the presence of internal nick sites because of the dynamic nature of rapid large DNA amplification.
  • This dynamic aspect means that at any one time, some internal nick sites will be un-nicked. This is because although nicking is believed to occur at all nick sites, nicking does not occur at the same time. Therefore, the polymerase synthesis, which proceeds to the next nicked site generates a variety of different sized DNAs.

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Abstract

L’invention concerne des procédés d’amplification rapide d’ADN volumineux englobant l’amplification du génome. Le procédé utilise des nickases et des polymérases mais pas d’initiateur. L’amplification d’un ADN volumineux peut se dérouler en moins de 10 minutes environ comme cela est déterminé par des bandes détectables de gels en commençant par 25 ng d’ADN dans un volume de 50 µl. Un avantage de ces procédés d’amplification d’ADN volumineux réside dans le fait que la séquence est représentée de manière proportionnelle. Les procédés peuvent également permettre d'obtenir un ADN amplifié de manière relativement rapide. L’amplification à base de nickase peut être complétée par une amplification à base d’initiateur pour augmenter la concentration de séquences cibles particulières. Les procédés peuvent également servir à obtenir un profil d’empreinte de l’ADN.
PCT/US2006/043014 2005-11-09 2006-11-03 Amplification de génome WO2007056173A1 (fr)

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US73522705P 2005-11-09 2005-11-09
US60/735,227 2005-11-09

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WO2007056173A1 true WO2007056173A1 (fr) 2007-05-18

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US20140343265A1 (en) * 2011-05-27 2014-11-20 Life Technologies Corporation Methods for manipulating biomolecules
CN114350756A (zh) * 2021-11-22 2022-04-15 西安交通大学 基于dna切刻/聚合链置换循环反应的全基因组自引发扩增方法及试剂盒

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US6191267B1 (en) * 2000-06-02 2001-02-20 New England Biolabs, Inc. Cloning and producing the N.BstNBI nicking endonuclease
WO2001090415A2 (fr) * 2000-05-20 2001-11-29 The Regents Of The University Of Michigan Procede de production d'une bibliotheque d'adn utilisant l'amplification positionnelle
US20030104431A1 (en) * 2001-07-15 2003-06-05 Keck Graduate Institute Nucleic acid amplification using nicking agents

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WO2001090415A2 (fr) * 2000-05-20 2001-11-29 The Regents Of The University Of Michigan Procede de production d'une bibliotheque d'adn utilisant l'amplification positionnelle
US6191267B1 (en) * 2000-06-02 2001-02-20 New England Biolabs, Inc. Cloning and producing the N.BstNBI nicking endonuclease
US20030104431A1 (en) * 2001-07-15 2003-06-05 Keck Graduate Institute Nucleic acid amplification using nicking agents

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140343265A1 (en) * 2011-05-27 2014-11-20 Life Technologies Corporation Methods for manipulating biomolecules
US11542535B2 (en) * 2011-05-27 2023-01-03 Life Technologies Corporation Methods for manipulating biomolecules
CN114350756A (zh) * 2021-11-22 2022-04-15 西安交通大学 基于dna切刻/聚合链置换循环反应的全基因组自引发扩增方法及试剂盒

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