WO2000049176A1 - Procede et appareil de separation de brin d'acide nucleique - Google Patents

Procede et appareil de separation de brin d'acide nucleique Download PDF

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Publication number
WO2000049176A1
WO2000049176A1 PCT/GB2000/000609 GB0000609W WO0049176A1 WO 2000049176 A1 WO2000049176 A1 WO 2000049176A1 GB 0000609 W GB0000609 W GB 0000609W WO 0049176 A1 WO0049176 A1 WO 0049176A1
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nucleic acid
ultrasound
dna
single strands
double stranded
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PCT/GB2000/000609
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English (en)
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David John Clarke
Nigel Peter Minton
Freidoun Zamani
Sarah Geraldine Fooks
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Microbiological Research Authority
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Priority to AU26801/00A priority Critical patent/AU2680100A/en
Publication of WO2000049176A1 publication Critical patent/WO2000049176A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/10—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing sonic or ultrasonic vibrations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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

  • the present invention relates to separation of nucleic acid strands, in particular to separation of double stranded DNA into single strands. More particularly, the present invention relates to amplification of DNA and to separation of DNA strands during DNA amplification.
  • a double-stranded DNA molecule can be considered as a stack of pairs of planar and rigid nucleotides. Due to the differential placement of hydrogen bond donor and acceptor groups, nucleotides have unique structural identities which in natural DNA only allow the specific pairings of adenosine with thymidine, and guanosine with cytosine.
  • the stack of nucleotides in a DNA strand is connected into a linear chain by a negatively charged phosphate backbone. In a double-stranded DNA molecule these chains assume the well-known double helical configuration described by Watson and Crick. It is the stacking and hydrogen bonding forces between the constituent nucleotides which hold the DNA double helix together.
  • the complementarity of the component strands of the double helix allows the construction of an exact copy of the original molecule on each of the strands after they have been separated.
  • the stacking interactions are mediated by van der Waal's forces (dipole- dipole interactions) and hydrophobic forces between the stacked nucleotides.
  • the stabilising energies of stacking and the hydrogen bonding between nucleotides in a base pair confer great stability on the DNA molecule. Additional stability is conferred by solvation.
  • the base stacking hydrogen bonding and solvation forces must be overcome.
  • Extremes of pH ⁇ 2 or > 1 2 2) causes ionisation of the nucleotide bases, loss of hydrogen bonding, and consequent strand separation. It is also well known that heating will disrupt both hydrogen bonds and the hydration shell, leading to the loss of forces holding the strands together
  • the temperature at which 50% of the DNA sample is single stranded is called the melting temperature (TJ
  • PCR polymerase chain reaction
  • strand separation In operation of PCR it is also essential that strand separation be reversible, in the sense that a single strand that has been separated from its hybridizing antisense strand can, under the right conditions, rehyb ⁇ dize with that antisense strand, or any other hybridizing strand.
  • elevated temperature separates DNA into single strands, and then at reduced temperature the single strands form double strands, or hybridize with a primer for synthesis of new nucleic acid
  • a disadvantage of current PCR techniques is that the reactants must be stable at the temperature required for separation of DNA strands, typically around 90-95 °C After strand separation, primer binding occurs at a lower temperature, typically 40-55 ° C and DNA synthesis at 65-72 ° C
  • PCR requires thermal cycling over a wide temperature range
  • thermal cycling must be rapid and requires high specification equipment.
  • Ultrasound is the periodic compression and rarefaction of fluids at frequencies exceeding the upper limit of the normal human hearing range ( 1 6000 Hz) and reaching to more than 500 MHz.
  • the range between 20 and 1 00 kHz is usually denoted the "power" range and frequencies above that regarded as “diagnostic” ultrasound .
  • the reason for this distinction is that much greater sound energy can be transmitted into a system at the lower frequencies.
  • Cavitation is the production of micro-bubbles in a fluid under the application of a large negative pressure. The bubbles are formed when the negative pressure exceeds the tensile strength of the water molecules. Theoretically the negative pressure must be more than 1 00 MPa, if no allowance is made for water vapour being formed within the bubble. In practice, cavitation occurs at very much lower acoustic intensity due to the unavoidable impurities and weak points in most liquids.
  • Cavitation is known to cause double-strand breakage of DNA and breakdown of the nucleotide bases, a property exploited in WO-A-93/031 50 and FR-A- 2654000.
  • WO-A-93/031 50 describes a method for simultaneous shear and denaturation of nucleic acids.
  • An aqueous mixture of nucleic acid and a chaotropic agent is subjected to a sufficient level of ultrasonic energy to produce cavitation in the mixture, thereby inducing breaks in long nucleic acid fragments so as to form shorter fragments.
  • the method is useful for lysing cells so as to release nucleic acids and other cell contents.
  • the mixture of nucleic acid and chaotropic agent is sonicated, typically at about 60 KHz.
  • FR-A-2654000 also uses ultrasound, or high frequency electromagnetic waves, to form a plasma around a medical sample or medical apparatus, causing breakdown of cell walls, denaturation of DNA and destruction of microbial contamination . Thus, all cell components are destroyed so that the medical sample or apparatus can be completely disinfected .
  • a first aspect of the invention provides a method of manipulation of a double stranded nucleic acid, comprising irradiating the nucleic acid with ultrasound so as to separate the nucleic acid into single strands along at least part of its length, substantially without strand breakage.
  • a double stranded nucleic acid is irradiated with ultrasound of sufficient power to separate the nucleic acid into the single strands.
  • the ultrasound power is controlled so that strand breaks substantially are not introduced into the nucleic acid .
  • a double stranded nucleic acid such as DNA
  • the strand separation is thus reversible and upon removal of or reduction in power of the ultrasound, the strands can hybridise to reform a double stranded nucleic acid, or can hybridise each with different single strands, such as PCR primers, so as to form double stranded nucleic acids.
  • a further advantage of the invention is that strand separation occurs below the high temperatures required using prior art PCR methodologies.
  • the invention thus enables the avoidance of high temperatures, hitherto routine in the PCR field. Whilst a range of thermostable enzymes have been identified for use in PCR, it is observed that after many cycles of heating and cooling, these enzymes nevertheless become denatured to a certain degree and lose their efficacy. Separation of a double sided nucleic acid into single strands without extreme thermal cycling thus offers the prospect of increased enzyme life in PCR methods.
  • the invention further opens PCR methods to the use of non-thermostable enzymes.
  • a preferred embodiment of the invention comprises irradiating the nucleic acid with ultrasound so as to separate the nucleic acid into single strands, and subsequently allowing single strands to hybridise to form a double stranded nucleic acid .
  • the method of the invention is suitable for separation and hybridisation of double stranded DNA, double stranded RNA, and a nucleic acid comprising a single strand of DNA and a single strand of RNA.
  • the ultrasound used in the invention is preferably chosen so that any heating of the nucleic acid solution that occurs is even throughout the solution, avoiding local areas of significantly increased temperature, referred to as "hot spots", which might damage DNA.
  • This is conveniently achieved by the use of ultrasound of relatively high frequency.
  • Ultrasound of frequency 200 KHz or higher is suitable and in particular ultrasound of frequency at least 500 KHz or higher. More particularly, it is convenient to use ultrasound having a frequency in the range 500 KHz to 3 MHz.
  • Ultrasound having increased frequency has a reduced wavelength and, in use, the spacing of standing waves within the nucleic acid solution is reduced compared to when ultrasound of shorter frequency is used. Build-up of local areas of high temperature is also reduced, thus tending to avoid associated damage and strand breakage to DNA.
  • PCR is a highly sensitive technique for amplification of nucleic acids and is of application to amplification of nucleic acids found at an extremely low level in a sample. Introduction of strand breaks into such a sample is undesirable if DNA in the sample is to be amplified by PCR.
  • a nucleic acid sample is irradiated with ultrasound of frequency about 1 .6 MHz, and complete separation of the nucleic acid into single strands is observed.
  • variation of the ultrasound power level between a power level at which the nucleic acid is separated into single strands and a power level at which single strands can hybridise, is used in a method for amplification of the nucleic acid. In this way, cycling of ultrasound power has been used to replace thermal cycling in PCR.
  • the prior art has described the desirability of breaking DNA strands by inducing cavitation in an aqueous mixture of nucleic acid. More precisely, the art describes inducing transient cavitation, in which gas bubbles formed in the mixture by ultrasound collapse violently. DNA damage and strand breakage is caused by the violent collapse of these bubbles.
  • transient cavitation in which gas bubbles formed in the mixture by ultrasound collapse violently. DNA damage and strand breakage is caused by the violent collapse of these bubbles.
  • Stable cavitation occurs when bubbles formed in solution oscillate about an equilibrium size at each cycle of ultrasound. These bubbles typically last for several ultrasound cycles and are distinct from the bubbles of transient cavitation which expand rapidly and collapse violently within the same acoustic cycle.
  • an amount of ultrasound is used that induces stable cavitation within the nucleic acid solution.
  • the ultrasound power level should not, however, be increased to the point at which transient cavitation and DNA strand breakage occurs.
  • a second aspect of the invention provides a method of separation of a double stranded nucleic acid into its component single strands, comprising irradiating the nucleic acid in solution with ultrasound with sufficient power to separate the nucleic acid into single strands along at least part of its length, but insufficient power to induce transient cavitation in the solution.
  • the ultrasound is conveniently at sufficient power to induce stable cavitation in the solution. Exposure to ultrasound over a long time period can result in localised increase in temperature in a solution of DNA, and so the method typically comprises use of high frequency ultrasound so that any consequent increase in temperature tends to be even throughout the solution. Another option is to maintain mixing of the solution sufficient to disperse such locally hot areas.
  • Ultrasound is of frequency 200 KMz or higher is suitable, though it is believed that any frequency may be suitable providing that conditions are controlled so as to avoid localised heating within the solution.
  • the ultrasound frequency is preferably 500 KHz or higher, more preferably 500 KHz to 3 MHz. In a specific embodiment described below, the frequency is in the range 1 - 3 MHz, and about 1 .6 MHz using a known ultrasound generator.
  • a further aspect of the invention provides apparatus for manipulation of a double stranded nucleic acid comprising
  • sonicating means for irradiating the nucleic acid with ultrasound of frequency 500 KHz or higher so as to separate the nucleic acid into single strands along at least a part of its length.
  • the reservoir optionally is for directly receiving a solution of nucleic acid into the apparatus, or for receiving a separate container such as a vial for holding a solution of DNA plus components, such as polymerase plus nucleoside triphosphates, suitable for synthesis of DNA according to PCR.
  • An apparatus of the invention conveniently comprises a large number of such reservoirs for individual such solutions.
  • the apparatus further comprises means for varying the ultrasound power.
  • a certain power level is required according to the invention to separate the nucleic acid into single strands. Once this is achieved, the ultrasound is turned off or at least reduced in power to a level at which either single strands can rehybridize, whether with the same original strand or another, or single strands can hybridize with PCR primer molecules for synthesis of new nucleic acid.
  • the sonicating means may be capable of generating ultrasound of frequency at least 200 KHz, more preferably 500 KHz - 3 MHz, though different frequencies are believed with the appropriate controls over cavitation in solution to be suitable also.
  • the apparatus may also comprise means for cooling the reservoir.
  • the cooling means may comprise means for circulating cooling liquid around the reservoir and means for deaerating the cooling liquid.
  • the invention thus provides apparatus for use in amplification of a nucleic acid analogously with PCR techniques, and suitably with the same reagents, but avoiding thermal cycling of the reaction mixture. PCR enzyme life may thereby be increased, as thermal cycling can damage such enzymes.
  • the method provides for the use of enzymes for PCR that would hitherto not have been suitable, being enzymes that are denatured or otherwise damaged by thermal cycling . It is known that certain enzymes are activated by or in the presence of ultrasound, and such enzymes may also now be of use in PCR according to the invention that includes use of ultrasound .
  • a further embodiment of the invention comprises separating a nucleic acid into single strands using a combination of both thermal cycling and ultrasound.
  • ultrasound strand separation and thermal strand separation are used in separate steps of the same amplification.
  • the invention in addition provides a method of amplification of a nucleic acid comprising
  • ultrasound separation of DNA is effectively used as a replacement for thermal denaturation of DNA. Cycles of separation and annealing are used in DNA amplification by PCR and the invention has successfully amplified DNA fragments of 3kb using ultrasound.
  • fragments up to about 4kb are suited to amplification using ultrasound, and the invention is also suited to amplification of smaller fragments of 2kb or less, or 1 kb or less in length.
  • the invention also comprises carrying out PCR using heat thermally to denature DNA and obtain a first PCR product and then carrying out PCR using ultrasound to separate DNA to obtain a second PCR product, this second product being the same length as or shorter than the first.
  • the method comprises irradiating the nucleic acid with ultrasound of sufficient power to separate it into single strands and of insufficient power to cause transient cavitation in the solution.
  • the method comprises continuously irradiating the nucleic acid with ultrasound, varying the power between a higher power at which nucleic acid is separated into single strands and a lower power at which single strands hybridize to form nucleic acid.
  • the ultrasound frequency is typically 200 KHz or higher, preferably 500 KHz or higher, as in other aspects of the invention.
  • the invention also provides use of ultrasound in separation of a double stranded nucleic acid into single strands, without strand breakage.
  • the invention still further provides use of ultrasound in reversible separation of a double stranded nucleic acid into single strands, in place of or in addition to use of elevated temperature in the polymerase chain reaction.
  • Fig. 1 shows a schematic view of apparatus of the invention
  • Fig. 2 shows an optimised PCR for pUCBM20-Block III insert
  • Fig. 3 shows strand separation thermally and by ultrasound
  • Fig. 4 shows strand separation thermally and by ultrasound
  • Fig. 5 shows effect of temperature on strand separation
  • Fig. 6 shows effect of DMSO on strand separation thermally and by ultrasound; and Fig. 7 shows strand synthesis after ultrasound mediated strand separation .
  • Fig. 1 shows an ultrasound irradiation device, shown generally as 1 , in which a sample tube 2 is mounted in a glass cooling jacket 3 having a water-flow inlet tube 4 and a water-flow outlet tube 5.
  • a sonicating device shown generally as 6 comprises a screw cap 7, an electrical connector 8, a spacer tube 9, and an embedded layer piezo disc 1 0.
  • a piezo-electric ceramic transducer in a stainless steel housing.
  • the transducer was fed by a 25 W radio frequency power amplifier (Wessex Electronics RC301 -25) connected to an electronic signal generator (Hewlett- Packard Function Generator 331 4A) .
  • the cooling water was de-aerated prior to each experimental run.
  • Plasmids SPApB3 and pUCBM20-Block III were grown and purified by conventional means. To make substrates suitable for ultrasonication experiments the supercoiled DNA products were converted to linear form by digestion with a restriction enzyme cutting once within each plasmid. The enzymes used were Bam ⁇ for SPApB3 and Nde ⁇ for pUCBM20-Block III. Each of these linear DNA molecules was about 3 kb in length.
  • Reaction mixtures were assembled in 50 ⁇ volumes and inserted into the glass jacket and the temperature probe positioned above it. Cooling water at a known temperature was circulated by means of a pump. The time and intensity of ultrasound irradiation was controlled manually in this prototype apparatus, and the temperature of the reaction mixture noted at regular intervals. Power was defined by the input voltage to the power amplifier and measured in millivolts. The ultrasound power level is approximately proportional to the square of the input voltage (maximum 25 Watts), and power was kept below the level at which transient cavitation might have occurred.
  • Electrophoretic analysis of DNA was performed in 1 % agarose gels cast in TAE buffer (40 mM Tris-acetate pH 8.3, 1 rriM EDTA) containing 0.5 ⁇ g ethidium bromide per ml Under these conditions single-stranded DNA is clearly distinguishable from duplex DNA of the same length by its greater mobility.
  • Primer elongation on a single-stranded DNA template was detected via the incorporation of fluoresce ⁇ n- 1 1 -dUTP, using reagents provided with the Gene Images labelling kit of Amersham International.
  • Reaction mixtures including DNA template (purified Block III PCR product, see above) , primers (as used for PCR above), deoxynucleoside t ⁇ phosphates and buffer components were assembled in a volume of 50 ⁇ l. They were then subjected to ultrasound or thermal (94°, 2 mm) strand separation procedures, and then chilled on ice. Enzyme (Klenow fragment of DNA polymerase; 5 U) was added and the reaction incubated at 37 ° for 2 h.
  • Plasmid DNA Strand Separation Initial experiments were designed to investigate the conditions required for ultrasound-mediated DNA strand separation, and to provide an unequivocal demonstration of the phenomenon. In these experiments plasmid DNA was used as the target. To permit the ready detection of strand separation, plasmid DNA was linearised by cutting at a single site to give a linear double- stranded DNA molecule about 3 kb in length. In Fig.
  • Lanes M shown 1 kb markers, lane 1 is untreated ds DNA, lane 2 is thermally-denatured DNA (94°C, two minutes)
  • Lanes 3-5 were irradiated using an input voltage of 1 50 V for two minutes (maximum temperature 26 °C)
  • lanes 6-8 had an input voltage of 200 mV, for two minutes (maximum temperature 32 °C)
  • lanes 9 and 1 0 had an input voltage of 250 mV, for two minutes (maximum temperature 37 ° C)
  • lanes 1 1 and 1 2 had an input voltage of 300 mV, for two minutes (maximum temperature 51 ° C) .
  • DNA is N ⁇ -/e/-linearised pUCBM20-Block II plasmid DNA and lanes M show 1 kb markers
  • lane 1 is untreated ds DNA
  • lane 2 is thermally-denatured DNA (94°C, two minutes)
  • lanes 3-5 had an input voltage of 300 mV, for 30 seconds (maximum temperature 68 °C)
  • lanes 6-8 had an input voltage of 300 mV, for 60 seconds (maximum temperature 63 °C)
  • lanes 9- 1 1 had an input voltage of 250 mV, for 30 seconds (maximum temperature 45 °C) .
  • lane M is 1 kb markers
  • ds is double stranded DNA
  • ss is single stranded DNA, denatured at 94°C for two minutes. The temperatures are indicated on the figure.
  • DMSO semi-polar solvent dimethyl sulphoxide
  • lanes 9- 1 1 show 1 0% DMSO (maximum temperature 64°C).
  • the next step in determining the utility of this process was to demonstrate that single-stranded DNA generated by this procedure was capable of acting as template for the elongation of a complementary primer i.e. for the resynthesis of the complementary DNA strand.
  • the substrate chosen for this was double- stranded DNA of about 0.5 kb generated by conventional PCR from pUCBM20-Block HI DNA using the universal and reverse universal primers, which were complementary to opposing DNA sequences flanking the insert.
  • this DNA was denatured thermally or with ultrasound in the presence of these primers, and dNTPs including fluorescein-labelled dUTP. After addition of DNA polymerase (Klenow fragment) resynthesis of complementary strands would be evidenced by fluorescein-labelled duplex DNA of the same size as the original PCR product. Such resynthesis would be dependent upon the presence of single-stranded DNA to act as template for this process.
  • Fig. 7 shows second strand resynthesis after ultrasound-mediated strand separation.
  • PCR-amplified III DNA was exposed to strand-separating conditions, and primer elongation followed by fluorescein-dUTP incorporation. Newly synthesised DNA was detected by a fluorescent.
  • Lanes 1 -3 show ultrasound strand separation
  • lane 4 shows thermal strand separation
  • lanes 5 and 6 show no strand separation
  • lane 7 shows no DNA
  • lane 8 shows no DNA polymerase.
  • Fig. 7 shows that such resynthesis does in fact occur, both after ultrasound irradiation (lanes 1 -3) and after thermal denaturation (lane 4) . Importantly, in the absence of measures to provide single-stranded DNA template, no labelled DNA of the expected size is produced (lanes 5 and 6) .
  • the template was bUCBM 20-block III DNA (Ndel - linearised). Separate lanes (not shown) were used for 1 kb markers, no amplification, no template with 35 thermal cycles, 1 5 thermal cycles, 35 thermal cycles, 20 ultrasound cycles and 1 5 thermal cycles. Again, power levels were such as to avoid cavitation in the mixture. Detectable DNA product results from 35 thermal PCR cycles, but not from only 1 5 cycles. However, if 1 5 thermal PCR cycles are preceded by 20 rounds of ultrasound-mediated strand separation and resynthesis, DNA product of the expected size is detected. Thus this experiment demonstrates that successive rounds of ultrasound-mediated strand separation did lead to detectable amplification of DNA, and also that non-linearised plasmid DNA is amenable to strand separation by this means. We have thus been successful in repeating this experiment.
  • a possible explanation for the strand separation effect is the hydrophobicity of small bubbles in water. Air bubbles in water carry some excess electric charge. The repulsive effect of this charge is the reason that stable bubbles in water can exist. It can be hypothesised that if these bubbles are small enough, they can migrate to the hydrophobic parts of the DNA and disrupt both the hydrogen bonds and the stacking force to the point at which the polymer will melts even at the ambient temperature, though we do not wish to be bound by this theory.
  • the complementary strands of double- stranded DNA molecules can be reversibly separated from each other by ultrasonic irradiation under appropriate conditions.
  • This strand separation has so far been demonstrated on linear DNA up to about 3 kb in length, takes place at moderate temperatures without significant strand breakage, and results in products capable of acting as templates in primer elongation reactions. Successive rounds of such strand separation and resynthesis result in DNA amplification. It has been shown that ultrasonic strand separation can mimic the thermal strand separation used in conventional PCR processes. This invention is potentially capable of facilitating the development of further diagnostic and other processes requiring DNA amplification or synthesis, or separation of double stranded DNA into single strands.

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Abstract

Un acide nucléique, généralement l'ADN, est irradié avec des ultrasons, de manière à le séparer en brins simples sur une partie de sa longueur, et sans occasionner de rupture des brins. Cette étape de séparation par ultrasons est également utilisée lors d'un procédé de réaction en chaîne de polymérase à la place d'une étape de chauffe, afin que des cycles rapides de séparation et de synthèse d'ADN se produisent sans cycle thermique.
PCT/GB2000/000609 1999-02-19 2000-02-21 Procede et appareil de separation de brin d'acide nucleique WO2000049176A1 (fr)

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