WO2012145449A1 - Procédés et dispositifs pour amplifier des acides nucléiques - Google Patents

Procédés et dispositifs pour amplifier des acides nucléiques Download PDF

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Publication number
WO2012145449A1
WO2012145449A1 PCT/US2012/034154 US2012034154W WO2012145449A1 WO 2012145449 A1 WO2012145449 A1 WO 2012145449A1 US 2012034154 W US2012034154 W US 2012034154W WO 2012145449 A1 WO2012145449 A1 WO 2012145449A1
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nucleic acid
dna
electrical field
amount
annealed
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PCT/US2012/034154
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English (en)
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Raul Cuero RENGIFO
Juliana Maria NAVIA
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International Park Of Creativity
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • PCR technology which is typically used in the amplification of DNA, is based on the polymerase chain reaction.
  • a test tube system for DNA replication allows a "target" DNA sequence to be selectively amplified or enriched several fold in several hours.
  • high temperature is used to separate the DNA molecules into single strands (one to several minutes at 94-96 °C), and synthetic sequences of single- stranded DNA (20-30 nucleotides) serve as primers. Two different primer sequences are used to bracket the target region to be amplified.
  • the primer anneals to the DNA by way of hydrogen bonds.
  • the annealing step occurs from one to several minutes at 50-65 °C.
  • the DNA is subsequently heated from one to several minutes at 72 °C in the presence of a polymerase, during which time the polymerase binds and extends a complementary DNA strand from each primer.
  • the methods generally involve exposing the nucleic acid to a mini-current electrical field while performing the steps of PCR.
  • the methods provide numerous advantages over current PCR techniques such as reduced reaction times, no heating requirements, and reduced amounts of reagents (e.g., reduced amounts of polymerase). Additionally, the methods described herein require significantly shorter reaction times (e.g., 35 minutes) compared to conventional PCR techniques (minimum 2 hours). Finally, as shown in the Examples below, the methods described herein amplify more DNA compared to conventional PCR techniques. In summary, the methods and devices described herein provide a more efficient and cost-effective way to perform PCR when compared to current PCR techniques.
  • Figure 1 shows an example of an electrolysis cell for performing the methods described herein.
  • Figure 2 shows electrophoresis gel bands of PCR samples from pYES vector after being run by electrolytic micro current PCR system (EMPS) described herein compared to conventional temperature-based PCR (CTP).
  • EMPS electrolytic micro current PCR system
  • Figure 3 shows electrophoresis gel bands from DNA amplifications of the pBSK coiled plasmids at different concentrations by EMPS and CTP.
  • Figure 4 shows electrophoresis gel bands of linearized plasmids amplified by EMPS as compared to CTP.
  • Figure 5 shows electrophoresis gel bands of genomic DNA amplified by EMPS as compared to CTP.
  • Figure 6 shows electrophoresis gel bands of cDNA amplification of RNA by EMPS as compared to CTP.
  • Figure 7 shows E.coli colonies grown in SOB plus ampicillin media after transformed with plasmids amplified by EMPS holding different metal sequences.
  • Figure 8 shows electrophoresis gel bands of digested plasmids that have been previously amplified by EMPS.
  • Figure 9 shows electrophoresis gel bands of calcium and sodium inserted in their respective vectors after run by both EMPS and CTP sequentially.
  • Figure 10 shows an example of a device for performing real time PCR using the methods described herein.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent of a component is based on the total weight of the formulation or composition in which the component is included.
  • the methods generally involve exposing the nucleic acid to a mini-current electrical field while performing the steps of PCR. In one aspect, the method involves:
  • the nucleic acid can be any molecule where it is possible and desirable to amplify (i.e., generating multiple copies of a specific nucleic acid sequence).
  • the nucleic acid can be an oligonucleotide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA).
  • the nucleic acid of interest introduced by the present method can be a nucleic acid from any source, such as a nucleic acid obtained from cells in which it occurs in nature, recombinantly produced nucleic acid, or chemically synthesized nucleic acid.
  • the nucleic acid can be cDNA or genomic DNA or DNA synthesized to have the nucleotide sequence corresponding to that of naturally-occurring DNA.
  • the nucleic acid can also be a mutated or altered form of nucleic acid (e.g., DNA that differs from a naturally occurring DNA by an alteration, deletion, substitution or addition of at least one nucleic acid residue) or nucleic acid that does not occur in nature.
  • the DNA can be genomic DNA.
  • the DNA can be double- stranded DNA including, but not limited to, a plasmid (linear or coiled, etc.), cosmid, phage, viral, YACS, BACS, other artificial chromosomes, and the like).
  • the DNA can be single stranded.
  • the nucleic acid can be a functional nucleic acid.
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
  • Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting.
  • functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, siRNA, miRNA, shRNA and external guide sequences.
  • the functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
  • Functional nucleic acids can be a small gene fragment that encodes dominant-acting synthetic genetic elements (SGEs), e.g., molecules that interfere with the function of genes from which they are derived (antagonists) or that are dominant constitutively active fragments (agonists) of such genes.
  • SGEs can include, but are not limited to, polypeptides, inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys, and small peptides.
  • SGEs can include, but are not limited to, polypeptides, inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys, and small peptides.
  • the small gene fragments and SGE libraries disclosed in U.S. Patent Publication No. 2003/0228601 which is incorporated by reference, can be used herein.
  • the methods described herein involve the application of a mini-current electrical field to a sample containing the nucleic acid and primer in the absence of heat in order to amplify a nucleic acid sequence of interest.
  • the reaction is performed less than or equal to 30 °C, preferably less than or equal to 25 °C.
  • the amount and duration of the electrical field applied to the nucleic acid can vary.
  • the amount of electrical field applied in steps (a) and (b) above can be the same or different value.
  • the duration of exposure to the electrical field can be the same or different in steps (a) and (b).
  • Different voltages can be used including different sets of micro currents.
  • the current is 50 mV/25 mA, 100 mV/50 mA, 150 mV/80 mA, 200 mV/100 mA, 250 mV/100 mA, 300 mV/150 mA, 500 mV/250 mA, 800 mV/400 mA, 1000 mV/500 mA, 1500 mV/800 mA, or 2000 mV/1000 mA.
  • the amount of the electrical field in steps (a) and (b) is from 50 mV to 2,000 mV and from 25 mA to 1,000 mA.
  • the amount of the electrical field in steps (a) and (b) is from 800 mV to 1 ,000 mV and from 400 mA to 500 mA.
  • the duration of exposure in steps (a) and (b) can range from 5 minutes to 60 minutes, 5 minutes to 50 minutes, 5 minutes to 40 minutes, 5 minutes to 30 minutes, or 5 minutes to 20 minutes.
  • the sample of nucleic acid can be prepared using techniques known in the art for preparing samples used in conventional PCR techniques.
  • the nucleic acid can be dissolved in water and buffer, where the buffered solution contains a divalent cation such as Ca +2 or Mg +2 .
  • the primer can be added directly to the sample containing the nucleic acid. This is not the case with conventional PCR.
  • the first step in conventional PCR involves the denaturing of the DNA by heat in the absence of the primer. It is only after the DNA is denatured that the primer is added to the sample followed by a second heating cycle.
  • the methods described herein are more efficient compared to conventional PCR, where an additional step is required prior to annealing the primer to the DNA.
  • the method involves:
  • the primers useful herein can be an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer should be sufficiently long to prime the synthesis of extension products in the presence of the polymerase.
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain more or fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to the different strands of each specific sequence to be amplified. This means that the primers should be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. However, for detection purposes, particularly using labeled sequence-specific probes, the primers typically have exact
  • a polymerase is added to the sample containing the annealed nucleic acid, and the sample is exposed to the mini-current electrical field for a sufficient time and duration in order to extend or elongate the sequence of interest.
  • Polymerases are enzymes that
  • RNA polymerase of the types I-V can be used herein.
  • RNA polymerases such as type I-III and T7 RNA polymerase can be used.
  • Another advantage of the methods described herein is that lower amounts of polymerase are required compared to conventional PCR techniques. For example, the methods described can use 0.3 ⁇ lL or 0.5 ⁇ lL of polymerase. Thus, in one aspect, the amount of polymerase used herein is up to 50% or 70% less than the amount needed in conventional PCR techniques.
  • the methods described herein can be used in any application where conventional PCR is used.
  • the methods described herein permit the isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA.
  • hybridization probes for Southern or northern hybridization and DNA cloning which require larger amounts of DNA, can be produced by the methods described herein.
  • the methods described herein can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. Any type of organism can be identified by examination of DNA sequences unique to that species.
  • Applications of the methods described herein with respect to forensics include, but are not limited to, identifying potential suspects whose DNA may match evidence left at crime scenes, exonerating persons wrongly accused of crimes, identifying crime and catastrophe victims, establish paternity and other family relationships, identifying endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers), detecting bacteria and other organisms that may pollute air, water, soil, and food, match organ donors with recipients in transplant programs, determine pedigree for seed or livestock breeds, authenticate consumables such as caviar and wine, and analyze ancient DNA that is tens of thousands of years old.
  • PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely.
  • PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a very high sensitivity.
  • the methods described herein can be used as an analytical tool to evaluate the content and purity of nucleic acids when deigning and synthesizing new drugs.
  • the methods described herein can substitute conventional PCR in any application related to drug design and analytical evaluation thereof.
  • the methods described herein can be used to amplify and simultaneously quantify a target nucleic acid in real time (i.e., quantitative real time polymerase chain reaction or qPCR).
  • the detection of target DNA can be performed by the use of (1) non-specific fluorescent dyes that intercalate with any double- stranded DNA, and (2) sequence- specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.
  • the methods described herein are performed in an electrolysis cell (20 in Figure 1).
  • the electrical field is produced inside the electrolysis cell, where the sample of nucleic acid is placed in a PGR tube 22 (Axygen, Inc.).
  • the volume of the sample tube can vary from 0.2 mL, 0.6 mL, 1.0 mL, 1.5 mL and 2.0 mL.
  • the thickness of the tube wall can also vary from 0.5 mm to 1.5 mm, preferably 1.15 mm to 1.20 mm, more preferably, 1.17 mm.
  • the thickness of the sample tube can be varied in order to maximize electrical current distribution.
  • the electrolysis cell can have a number of different dimensions.
  • the cell is 20 x 20 x 10 cm, 15 x 15 x 10 cm, 10 x 10 x 5 cm, 10 x 10 x 10 cm, 5 x 5 x 5 cm, 10 x 10 x 10 cm; 20 x 15 x 10cm, 15 x 13 x 12 cm, or 10 x 5 x 5 cm.
  • the cells can be different shapes including, but not limited to, round, rectangular, and square.
  • the housing of the cell can be composed of different materials including, but not limited to, plastic, rubber, pure glass, mixed glass or Plexiglas, acrylic and polycarbonate.
  • the cell can also be used with an optional lid 23 so that the cell is a closed system. In certain aspects, it is desirable to use a lid in order to avoid sample evaporation or contamination.
  • the cell can be designed to hold as many sample tubes as needed.
  • the cell In order to establish an electrical field in the cell, the cell is divided into two compartments (see Figure 1), one representing a cationic field 24 and the other representing an anionic field 25.
  • a synthetic membrane 26 was used to divide the electrolysis cell in two
  • the electrolysis cell is connected to a power supply 27 (e.g., EC- 105 Electrophoresis Power Supply by Thermo EC. (Inv #8298)) in the external end and to the membrane in the electrolysis cell.
  • a power supply 27 e.g., EC- 105 Electrophoresis Power Supply by Thermo EC. (Inv #8298)
  • the two compartments are filled with electrolyte solution for current conduction.
  • Different types of electrolytes can be used herein, including sulphuric acid, hydrochloric acid, iron solution, potassium chloride, and Tris-Running buffer TAE. Additionally, different concentrations of the electrolyte can be used (e.g., IX, 2X, 5X, 10X and 20X).
  • Each compartment of the cell has a different electrolyte concentration.
  • the working compartment where the samples of nucleic acid are placed, has the higher concentration of electrolyte (cationic compartment in Figure 1), while the reference compartment (anionic compartment in Figure 1) had the lower concentration of electrolyte.
  • Various concentration ratios of the buffer between the cells can be used. In one aspect, the ratio of buffer concentration in the working compartment relative to the reference compartment is 1 : 1 2: 1 3: 1 1.5: 1 2.5: 1 and 4: 1.
  • the electrolysis cell can be closed with a lid, hermetically, thus preventing outside penetration of air.
  • the lid covering the electrolysis cell has two openings where the working electrode (e.g., cationic 28) and the reference electrode 29 (e.g., Ag/AgCl reference electrode, double junction, SGJ, Metrohm plug-in head B) were passed through and connected to the power supply in order to establish the electrical field.
  • Samples containing the nucleic acid are next placed inside the electrolysis cell and submerged into the buffer inside electrolysis cell until sample volume inside the vial reached electrolyte solution inside the cell box.
  • the vials 22 can be held by a wooden or plastic support 21 attached to one of the box walls in order to keep them upright.
  • the vials can be submerged completely in the buffer because they are completely sealed.
  • voltage and amperage are applied in order to expose the samples to an electrical field.
  • the electrical field generated in the cell can be measured by the use of voltmeter 30, which is attached to the mesh electrode 31 via wires 32.
  • the mesh electrode 31 is attached to the synthetic membrane 26 in Figure 1.
  • Figure 10 provides another exemplary device for performing the methods described herein, particularly real time PCR.
  • the sample support 1 has a dark bottomless area for qPCR and RealTime analysis.
  • the electrodes 2 (working and reference) generate and maintain the electrical field in solution.
  • a programmable electrical and digital system 3 applies voltage and mini currents to the system and is connected to a power supply.
  • An adaptable optical system 4 reads sample fluorescence for qPCR and RealTime analysis.
  • Components 1-4 are integrated parts of the system attached to the lid 11 that fits and matches the bottom holder-area 12 supporting the samples vials and seals the cell.
  • the electrolysis cell compartment is divided into two
  • compartments (10 in Figure 10 is one compartment) by a middle divider 5 composed of a silver mesh 13 and an ionic membrane 9.
  • Silver wires 7 are connected to the power supply and divider in order to produce a mini current through the electrical cell system.
  • a multi-meter 8 measures the voltage and current applied.
  • the optical device useful for qPCR and RealTime analysis includes a light source of emission, a specific filter within 450 and 520 nm of wave length, and a detection system designed to couple to the system to read fluorescence produced by probes such as, for example, SYBRgreen or taqman probes present in the sample vials prior to exposing the sample to the electrical field.
  • the system can be adapted to read each sample independently during the during amplification process over time.
  • the bottom of the cell can be configured with dark walls in order to avoid interference of media light and from side samples. Measurements can be taken during the amplification process, and the detector transducer can change the signal into a fluorescence value that is correlated with a standard curve to determine number of copies produced during the amplification process.
  • the methods described herein provide numerous advantages over current PCR techniques such as the use of lower amounts of reagents (e.g., reduced amounts of polymerase). Additionally, the methods described herein require significantly shorter reaction times (e.g., 35 minutes) compared to conventional PCR techniques (minimum 2 hours). The fact that the methods require no heat and shorter reaction times means there is less opportunity for degradation and formation of side -products. Indeed, as shown in the Examples below, the methods described herein amplify more DNA compared to conventional PCR techniques. As discussed above, conventional PCR requires controlled heating steps at every stage, which adds to the overall cost and inefficiency of the amplification process. Finally, the methods described herein produce nucleic acids that are more pure than conventional PCR.
  • reaction conditions e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
  • a pYES plasmid vector holding a sequence of YDL194W gene of 2660 bp (Clontex) was amplified with designed primers to amplify the YDL194W gene sequence and backbone vector.
  • the sample had an initial concentration of 0.1 g/ l ( Figure 2).
  • FIG. 2 shows electrophoresis gel bands of PCR samples from pYES vector after being run by electrolytic micro current PCR system (EMPS) described herein compared to conventional temperature-based PCR (CTP).
  • the bands from the EMPS are more pronounced (lane 1 and lane 5) while CTP are less pronounced (lane 2 and 4). All wells were sampled with the same exact sample volume.
  • the bands in the gel indicate the following: Lane 1. Sample one (YDL194W gene) amplified by EMPS Lane 2. Sample one amplification trough standard method CTP. Lane 3. Molecular marker (1 Kb). Lane 4. Sample 2 (Pichia stipitis CBS 6054 (SNF3) gene) amplified trough standard CTP method. Lane 5. Sample 2 amplification trough new method EMPS.
  • Plasmid stocks were diluted until plasmid DNA concentrations of 0.005, 0.01, 0.015 and 0.02 ⁇ g/ ⁇ l were obtained.
  • Figure 3 shows electrophoresis gel bands from DNA amplifications of the pBSK coiled plasmids at different concentrations, after run by EMPS, compared to CTP.
  • the samples run by EMPS show higher amplification than the run by CTP even at lower concentration of DNA. All wells were sampled with the same exact sample volume.
  • the bands in the gel indicate the following: Lane 1. Molecular marker (1 Kb). Lane 2. Sample one (calcium plasmid with 0.005 ⁇ g/ ⁇ l starting concentration) amplified by new method EMPS. Lane 3. Sample one (calcium plasmid with 0,005 ⁇ g/ ⁇ l starting concentration) amplified by standard CTP. Lane 4.
  • Sample two (calcium plasmid with 0.01 ⁇ g/ ⁇ l starting concentration) amplified by EMPS. Lane 5. Sample two, (calcium plasmid with 0.01 g/ l starting concentration) amplification by CTP. Lane 6. Sample three (sodium plasmid with 0.015 g/ l starting concentration) amplified by new method EMPS. Lane 7. Sample three, (sodium plasmid with 0.015 ⁇ g/ ⁇ l starting concentration) amplified by CTP. Lane 8. Sample four, (Vanadium plasmid with 0.02 ⁇ g/ ⁇ l starting concentration) amplified by EMPS. Line 9. Sample four (Vanadium plasmid with 0.02 ⁇ g/ ⁇ l starting concentration) amplified by CTP.
  • Linear plasmid A pBSK plasmid containing sodium biding protein gene sequence (1934 bp) was linearized with a standard digestion protocol with Psil enzyme purchased from NEB, (New England Biolabs inc. R0657L) for 2.5 hours at 37 °C with alkaline phosphatase treatment. The final linearized plasmid concentration was 0.025 ⁇ g/ ⁇ l.
  • Sodium biding protein gene sequence was amplified with pUC/ml3 forward and reverse primers from Promega, which amplified the sequences between the vector cloning site. The target of approximate 2,000 bp was amplified.
  • Figure 4 shows electrophoresis gel bands of linearized plasmids amplified by EMPS as compared to CTP.
  • the linearized amplification shows that the EMPS maintains intact the nature of the DNA. No denaturing of DNA occurred. All wells were sampled with the same exact sample volume.
  • the bands in the gel indicate the following: Lane 1. Molecular marker (1 Kb) Lane 2 and Lane 4. Linearized plasmid holding sodium biding protein gene sequence amplified by EMPS. Lane 3 and Lane 5. Linearized plasmid holding sodium biding protein gene sequence amplified by CTP.
  • Genomic DNA of a Geobacullus spy.'. 18 hours pure culture was extracted and purified with QIAGEN DNeasy Blood & Tissue Total DNA purification kit. Extracted genomic DNA resulted in 0.072 ⁇ g/ ⁇ l of DNA concentration. Sample Amplification with random hexamers, from Promega CI 181 was carried out.
  • Figure 5 shows electrophoresis gel bands of genomic DNA amplified by EMPS as compared to CTP.
  • the bands from the EMPS are more pronounced than the CTP. All wells were filled with the same exact sample volume.
  • the bands in the gel indicate the following: Lane 1. Molecular marker (1 Kb). Lanes 2, 3, 6 and 7. Genomic Geobacillus spp. amplified DNA by EMPS. Lanes 4, 5, 8 and 9. Genomic Geobacillus spp. amplified DNA by CTP.
  • Polyadenylated RNA 0.1 g/ l (Promega cat# A3500) was initially reverse transcribed with QIAGEN Omniscript Reverse Transcription kit cat# 205113 for one hour at 37 ° C with reverse transcription reaction buffer. cDNA was amplified with oligodT primers from the same kit. A 1.2 KB fragment was amplified as expected.
  • Figure 6 shows electrophoresis gel bands of cDNA amplification of RNA by EMPS as compared to CTP. Intensified bands were observed by EMPS. All wells were filled with the same exact sample volume. The cDNA amplification shows that EMPS does not affect the nature of the DNA, thus no denaturation of DNA occurred.
  • the bands in the gel indicate the following: Lane 1. Molecular marker (1 Kb). Lane 2. cDNA amplified by CTP. An unexpected band of approximate 2,000 bp was observed. Line 3. cDNA amplified by EMPS. An expected band of approximate 1,200 bp produced.
  • the amplification process was performed using the device as shown in Figure 1 and the standard PCR thermocycler (Maxigene gradient thermo-1000 from Axygene). Enzymes and reagents used for master mix and reaction were from Promega. Magnesium Chloride Solution cat # A3511. dNTPs mix cat # C114B, Magnesium free buffer cat# M190G, Nuclease free water part# PI 19E and taq polymerase cat #M166B and DNA polymerase 1 cat # M205A, except for RT and cDNA, where the Quiagen Omniscript reverse transcription kit and TopTaqPCR master mix kit reagents were used for amplification.
  • Time sets for the 3 step process included 20/ 10 / 20 minutes, 5/ 5 /10 minutes, 15/ 10 / 20 minutes and 10/5/20 minutes, were tested, with 10/5/20 the preferred time schedule.
  • DNA polymerase and dNTPs mix were added to the samples to complete the amplification process.
  • the volume of DNA polymerase and dNTPs mix was less than or equal to the volume used in CTP (0.5 ⁇ or 0.3 ⁇ of polymerase and 2 or 1.5 ⁇ of dNTPs).
  • Samples were exposed to an electric filed of 900 mV and 450 mA for 20 minutes.
  • spectrophotometer GE nanospectrophotometer GE nanospectrophotometer. Tables 1-4 demonstrate that EMPS is able to amplify DNA as effectively if not better than standard PCR (CTP), which resulted in higher DNA concentrations.
  • CTP standard PCR
  • Example 1 DNA concentrations of two different samples, (samples 1 (YDL194W gene) and 2 (Pichia stipitis CBS 6054 (SNF3) gene) amplified by EMPS as compared to CTP.
  • sample 4 (sodium plasmid with 0.015 ⁇ g/ ⁇ l starting concentration) and sample 4 (Vanadium plasmid with 0.02 ⁇ g/ ⁇ l starting concentration) after amplification by EMPS, as compared to CTP. Even at lower concentrations of DNA the amplifications were higher with EMPS than the CTP.
  • SOB Lia Brethani plus salts
  • Figure 7 shows E.coli colonies grown in SOB plus ampicillin media after transformed with plasmids amplified by EMPS holding different metal sequences. Positive control was taken as same E. coli competent cells transformed with CTP amplified product. Higher number E. coli colonies transformed with plasmid amplified by EMPS as compare to the lower number of colonies transformed with plasmid amplified by CTP (positive control).
  • Linearized and amplified plasmids were used for digestion to demonstrate the DNA did not suffer any damage during amplification by EMPS.
  • Figure 8 shows electrophoresis gel bands of digested plasmids that have been previously amplified by EMPS.
  • the plasmid is still intact and ready to be used after previously being amplified by EMPS.
  • the bands in the gel indicate the following: Lane 1. Molecular marker (1 Kb). Lane 2. Sodium Na amplified plasmid by EMPS digested with Spel and EcoRI enzymes, sequences was extracted from plasmid. Lane 3. Sodium Na complete and undigested amplified plasmid by EMPS. Lane 4. Calcium Ca amplified plasmid by EMPS digested with BamHI and Clal enzymes. Lane 5. Calcium Ca amplified plasmid by EMPS digested with BamHI and Apal enzymes. Lane 6. Calcium Ca complete and undigested amplified by EMPS.
  • the amplified samples obtained by EMPS were compared to conventional standard PCR (CTP) to ensure DNA sample integrity.
  • CTP conventional standard PCR
  • Promega GOTaq Green master mix reagents were used.
  • the same temperature cycles used to amplify initial samples were used to amplify these last samples already amplified by EMPS.
  • the presence of same size DNA from this amplification process as well as for EMPS indicates DNA is being correctly amplified and is not being degraded.
  • Figure 9 shows electrophoresis gel bands of calcium and sodium inserted in their respective vectors after run by both EMPS and CTP sequentially. The DNA remains intact, as proven by
  • the methods described herein are able to amplify DNA up to 4.5 fold more than conventional PCR methods. These results were obtained within very short time (35 minutes), after several trials in which exposure to specific current during different experiments showed the exact time DNA samples needed to denature, anneal and elongate.
  • Different currents were used in each step of the amplification process ranging from 250 mv and 125 ma, to 500 mV and 250 mA, 800 mV and 400 mA, 900 mV and 450 mA, 1000 mV 500 mA and 1250 mV and 650 mA showing consistent results.
  • the best amplification results were obtained with the current of 900 mV and 450 mA.
  • the present invention showed effective amplification results for different kinds of samples, including linear and coiled plasmids with a starting DNA concentration of 5 ng/ ⁇ , genomic DNA amplification with random primers, and with cDNA amplification processes. Targets of different sizes were tested, all showing consistent amplification results.
  • Successful transformation cells with amplified DNA produced by the methods described herein were achieved as wells as effective digestion and further standard PCR amplification of the amplified product, which showed there was no DNA damage during the new amplification process.

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Abstract

Les procédés et les dispositifs ci-décrits permettent d'amplifier des acides nucléiques. De manière générale, les procédés consistent à exposer l'acide nucléique à un champ électrique à minicourant pendant la mise en œuvre des étapes de la PCR. Ces procédés offrent de nombreux avantages par rapport aux actuelles techniques de PCR tels que des temps de réaction réduits, pas d'obligation de chauffage, et des quantités réduites de réactifs (par ex., quantités réduites de polymérase). De plus, les procédés ci-décrits requièrent des temps de réaction significativement plus courts (par ex., 35 minutes) comparativement aux techniques de PCR classiques (minimum, 2 heures). Pour finir, comme illustré dans les Exemples ci-dessous, les procédés ci-décrits amplifient plus d'ADN que les techniques de PCR classiques. En résumé, les procédés et les dispositifs selon l'invention offrent une façon plus efficace et plus rentable d'effectuer une PCR comparativement aux actuelles techniques de PCR.
PCT/US2012/034154 2011-04-22 2012-04-19 Procédés et dispositifs pour amplifier des acides nucléiques WO2012145449A1 (fr)

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US9044729B2 (en) 2012-07-27 2015-06-02 International Park Of Creativity Methods and devices for electromagnetic amplification of nucleic acids
US10350573B2 (en) * 2016-04-29 2019-07-16 Saint-Gobain Performance Plastics Corporation Radiation curable system and method for making a radiation curable article
US11267944B2 (en) 2015-12-30 2022-03-08 Saint-Gobain Performance Plastics Corporation Radiation curable article and method for making and using same

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US11267944B2 (en) 2015-12-30 2022-03-08 Saint-Gobain Performance Plastics Corporation Radiation curable article and method for making and using same
US10350573B2 (en) * 2016-04-29 2019-07-16 Saint-Gobain Performance Plastics Corporation Radiation curable system and method for making a radiation curable article

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