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WO2008122843A2 - Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud - Google Patents

Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud Download PDF

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Publication number
WO2008122843A2
WO2008122843A2 PCT/IB2007/051259 IB2007051259W WO2008122843A2 WO 2008122843 A2 WO2008122843 A2 WO 2008122843A2 IB 2007051259 W IB2007051259 W IB 2007051259W WO 2008122843 A2 WO2008122843 A2 WO 2008122843A2
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WO
WIPO (PCT)
Prior art keywords
dense
reactants
inert liquid
density
pcr
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Ceased
Application number
PCT/IB2007/051259
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English (en)
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WO2008122843A3 (fr
Inventor
Luís André PONTES
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Individual
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Individual
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Priority to PCT/IB2007/051259 priority Critical patent/WO2008122843A2/fr
Publication of WO2008122843A2 publication Critical patent/WO2008122843A2/fr
Publication of WO2008122843A3 publication Critical patent/WO2008122843A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention describes novel compositions and methods for simplifying and improving the polymerase chain reaction, a procedure for amplifying specific nucleic acid sequences which finds broad use in the fields of genetics, molecular biology, cellular biology, analytical biochemistry, clinical chemistry, and forensic science.
  • DNA amplification by the PCR technique can aim innumerous objectives such as disease diagnoses or more academic goals such as genome sequencing and studies of genetic variability among populations and species.
  • the PCR is performed in an appropriate tube or vessel, usually a plastic one, in which a mixture of essential reactants and one or more chemical additives are laid, comprising a total volume of usually about 20-200ul.
  • the basic mixture comprises a polynucleotide polymerase, preferably a thermostable DNA polymerase, most preferably the DNA polymerase I from Thermus aquaticus (Taq polymerase, subject of U.S. Pat. No.
  • oligonucleotide primers usually 15 to 30 nucleotides long and composed of deoxyribonucleotides, a magnesium compound, usually MgCl.sub.2; an aqueous buffer, pH 8-9 at room temperature, usually also containing KCl but to which several diferent chemical additives can be added to enhance the reaction efficiency; and finally, an aliquot of the test sample containing, or possibly containing, the target DNA to be amplified.
  • DNA which is accomplished by heating the sample in temperatures usually between 90-95 0 C.
  • the temperature of the sample is then lowered to usually 55-65°C in order for the primers to bind to the target sample to which they are complementary in a Watson- Crick manner.
  • the polymerase enzyme then catalyzes the template dependent complementary addition of the nucleoside triphosphates, at a temperature usually between 70-75 0 C. These steps are repeated usually through 30-40 cycles resulting in the exponential increase of the original DNA segment copy number.
  • Hot start PCR methods that rely upon a physical barrier to temporarily separate one essential reactant from the PCR premix, as the one described in document U.S. PI. No. 5 411 876, utilizes paraffin as its basic or even only material.
  • the physical-chemical principle involved in these techniques is the change in the state of matter undergone by paraffin as it goes from a solid to a liquid state in the first heating step of the DNA amplification reaction.
  • the idea behind these methods is the one of a physical barrier that can be automatically removed by the intrinsic and necessary heating steps of the PCR technique.
  • the format of these techniques is far from optimal, mainly because of the type of material utilized.
  • the use of a substance that is solid at room temperature is precisely what creates the main technical problems of these hot start techniques, because a solid material has to be previously melted and later obstructs the pipette tips used to recover the sample after the reaction is over.
  • the present invention is based on the inventive principle that a liquid material can be used as a much more suitable physical barrier in hot start PCR methods by the exploration of another physical-chemical property, which is density.
  • a layer of a dense liquid, chemically inert and insoluble in water is placed inside a suitable PCR tube or vessel, on top of a PCR premix containing all but one essential reactant.
  • This separated reactant dissolved in a aqueous buffer, is then placed within the liquid layer, in a position apart from the premix where it is immobilized by the density of the liquid, which is equal to its own.
  • the liquid is an oil, preferably a silicone oil.
  • the precise moment in which mixing of the reactants occur is a function of the initial distance between then and of the descendent velocity of the aqueous drop containing the separated reactant.
  • the initial distance depends on the height of the liquid column used to separate them, which can be controlled by the volume of liquid and also by the type of vessel used. A proper volume, for instance, can easily assure the merge of reactants only after 95°C had been reached, providing an simple, inexpensive and efficient hot start PCR method.
  • a premix containing all but one essential reactant is placed in the bottom of a tube or vessel suitable for performing the PCR technique; then a layer of a chemically inert, water insoluble liquid, which in the preferred embodiment is a silicone oil with density between 0.96 g/ml and 1.2 g/ml and preferably equal to 1 g/ml, is placed over the premix, in a final volume usually between lOul and 500ul; finally a small aqueous drop, comprising a volume usually between IuI and lOul of an appropriate buffer containing the separated essential reactant, is placed inside the separating liquid barrier, near the liquid-air interface, which is the point of maximum distance from the premix in the bottom of the tube or vessel.
  • a chemically inert, water insoluble liquid which in the preferred embodiment is a silicone oil with density between 0.96 g/ml and 1.2 g/ml and preferably equal to 1 g/ml
  • the separated reactant can be any one of the essential PCR reactants, e.g. the magnesium compound thermostable, the DNA polymerase or any one or all of the nucleoside triphosphates, but in the preferred embodiment it is one or more of the primers utilized for amplifying the target DNA sequence.
  • the density of the liquid which preferably is silicone oil, is equal to or slightly superior to the density of the separated reactant aqueous solution and so the drop, at a constant room temperature, is immobilized in a position completely apart from the other reactants in the bottom of the tube or vessel.
  • the drop begins to accelerate downwards to the premix in the bottom of the tube or vessel finally merging with the other reactants at an elevated temperature, usually between 92-95°C.
  • the moment at which the merging occurs can be regulated by the volume of the liquid barrier used.
  • the process of the present invention was used to solve the problem of primer-dimmer accumulation, or in another words, it was tested for its ability to function as an efficient hot start PCR method.
  • the critical feature is to be able to first, completely separate the premix from one essential excluded reactant and second, to automatically promote the merge of these reactants at the most elevated temperature possible.
  • the present invention was compared to a state-of-the-art hot start PCR method that uses a chemically modified Taq DNA polymerase, which is inactive at low temperatures and that regain activity only after a previous incubation of 10-20 min at 95°C, and also against the more traditional PCR procedure using no form of hot start.
  • the DNA amplification reaction were aimed at the amplification of a specific DNA sequence of the tropical parasite Leishmania chagasi, which is the etiologic agent of the tropical disease known as visceral leishmaniasis.
  • a mixture of two silicone oils composed by 999 parts of a standard silicone oil (polydimethylsiloxane) and 1 part of a hydrophilic silicone oil, used as a tissue softener.
  • samples were amplified using the basic or traditional PCR protocol (no hot start); samples of the second group were amplified using the process of the present invention, by which one of the primers (in this example, the backward primer) was not included in the premix, but was placed in the oil layer, near the oil-air interface, in complete separation from the other reactants; and finally the samples of the third group were amplified by a state-of-the-art hot protocol using a chemically modified Taq DNA polymerase. All samples were recovered with 100 ul of the silicone oil mixture to as a control measure and also to prevent evaporation by heating.
  • the primers in this example, the backward primer
  • Leishmania chagasi DNA was purified quantified by spectrofotometry and amplified by 35 cycles with 30 seconds at 95°C for template denaturation, 30 seconds at 65°C for primer annealing and 15 seconds at 72°C for primer extension. Cycles were preceded by 5 minutes at 95°C to assure complete template denaturation and the reaction ended by a 2 minute final extension step. Polyacrilamide gel electrophoresis followed by silver staining was chosen for visualization of amplification results due to its superior detection capability over agarose electrophoresis systems.
  • Figure 2 shows the result of the comparison between the different PCR protocols.
  • Parasite DNA was amplified by either by the traditional PCR protocol, using normal Taq DNA polymerase and no kind of hot start (first group: lanes 1-3); by the hot start method provided by the process of the present invention, also with normal Taq DNA polymerase (second group: lanes 4-6); or finally, by the hot start method that uses a chemically modified Taq polymerase which is inactive in low temperatures and regains activity only after a pre-incubation step of 10-20 minutes at 95°C (third group: lanes 7-9).
  • the first two lanes of each group contains samples in which 50 pg of L. chagasi DNA was amplified in duplicate and the last lane of each group contains samples in which no DNA was introduced (negative samples).
  • the efficiency of the hot start method provided by the process of the present invention is equivalent to the efficiency of the 'gold standard' used, as seen by the equivalency in eliminating the primer- dimmers and in enhancing the amplification yield of the specific target, as seen by the greater intensity of the specific product bands, which for silver staining is proportional to the amount of DNA present (until a staining saturation point is achieved).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé et un composant permettant de renforcer l'amplification d'ADN obtenue par une réaction en chaîne par polymérase. Ce procédé présente une amélioration dans le procédé appelé PCR à démarrage à chaud à barrière dans lequel une couche de paraffine solide est utilisée pour séparer un ou plusieurs réactifs essentiels des autres réactifs nécessaires jusqu'à ce qu'une température plus rigoureuse soit obtenue. Dans la présente invention, la paraffine solide est substituée par un liquide qui crée un procédé PCR démarrage à chaud plus efficace et plus pratique, lequel tire profit pour sa fonction d'une propriété physique-chimique différente.
PCT/IB2007/051259 2007-04-10 2007-04-10 Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud Ceased WO2008122843A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IB2007/051259 WO2008122843A2 (fr) 2007-04-10 2007-04-10 Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2007/051259 WO2008122843A2 (fr) 2007-04-10 2007-04-10 Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud

Publications (2)

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WO2008122843A2 true WO2008122843A2 (fr) 2008-10-16
WO2008122843A3 WO2008122843A3 (fr) 2009-04-23

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PCT/IB2007/051259 Ceased WO2008122843A2 (fr) 2007-04-10 2007-04-10 Utilisation de barrières liquides pour effectuer une pcr à démarrage à chaud

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Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0515506B1 (fr) * 1990-02-16 2000-01-05 F. Hoffmann-La Roche Ag Ameliorations apportees a la specificite et a l'applicabilite de la reaction en chaine de polymerases
US6641998B2 (en) * 1997-10-10 2003-11-04 Stratagene Methods and kits to enrich for desired nucleic acid sequences

Also Published As

Publication number Publication date
WO2008122843A3 (fr) 2009-04-23

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