WO2008122843A2

WO2008122843A2 - Use of liquid barriers to perform hot start pcr

Info

Publication number: WO2008122843A2
Application number: PCT/IB2007/051259
Authority: WO
Inventors: Luís André PONTES
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-04-10
Filing date: 2007-04-10
Publication date: 2008-10-16
Anticipated expiration: 2009-10-10
Also published as: WO2008122843A3

Abstract

The present invention describes a method and a component to enhance the DNA amplification achieved by the polymerase chain reaction. The method presents an improvement in the so-called Barrier Hot Start PCR method in which a layer of solid paraffin is used to separate one or more essential reactant from the other necessary reagents until a more stringent temperature is achieved. In the present invention, the solid paraffin is substituted by a liquid material, which creates a more practical and efficient Hot Start PCR method that takes advantage of a different physical-chemical property for its function.

Description

Description USE OF LIQUID BARRIERS TO PERFORM HOT START PCR

[ 1 ] FIELD OF THE INVENTION

[2] 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.

[3] BACKGROUND OF THE INVENTION

[4] The PCR technique is well known through documents U.S. PI. No. 4 683 195, U.S.

PI. No. 4 683 202 e U.S. PI. No. 4 965 188, and consist in a process by which one or more DNA segments are copied or amplified millions of times. Through this process one can obtain sufficient genetic material for any subsequent application that might otherwise be extremely difficult or even impossible. It is one of the most important techniques in the biomedical field and its invention has been awarded with the Nobel Prize in chemistry and represents the most expensive patent ever sold. 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.

[5] 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. 4,889,818, incorporated herein by reference); all four common nucleoside triphosphates (dATP, dTTP, dCTP, and dGTP); a pair or more of single- stranded 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.

[6] The amplification reaction begins with the separation of the double-stranded target

DNA, which is accomplished by heating the sample in temperatures usually between 90-95⁰C. 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 70-75⁰C. These steps are repeated usually through 30-40 cycles resulting in the exponential increase of the original DNA segment copy number.

[7] The practical advantages of PCR nucleic acid amplification have been rapidly appreciated in many fields of biomedical activities since it can replace a large fraction of molecular cloning and mutagenesis operations commonly performed in bacteria, having advantages of speed, simplicity, lower cost, and sometime increased safety. Besides that, PCR allows for the rapid and highly sensitive qualitative and quantitative analysis of nucleic acid sequences, without the use isotopic detection.

[8] Although it has represented a true revolution for the scientific research in the fields of biomedical sciences and also for the pharmaceutical and biotechnological industries, the classical PCR protocol presented some limitations that motivated the introduction of several technical improvements and variations.

[9] It is well know that one of the limitations of the original protocol is the generation of non specific products, which can vary in nature but is often the result of an inter-primer binding and the subsequent enzymatcly catalyzed nucleotide addition. These byproducts consume reactants that otherwise would be used in the amplification of the target sequence and so they lead to a reduction in the overall efficiency of the reaction.

[10] Many technical improvements were invented in order to solve the problem of non specific amplification and specifically those derived from primer-dimmer formation. Since these products are first generated in low temperatures mainly during sample preparation at room temperature, all these innovations work by delaying the reaction start until more elevated temperatures had been reached. Because of this common goal these techniques are collectively known by the popular name of 'hot start' PCR methods.

[11] One of these methods is described in document U.S. PI. No. 5,773,258. In this invention, a chemically modified DNA Taq polymerase which is provided in an inactive formulation is used in place of the normal Taq polymerase. This modified enzyme only regains activity after a pre-incubation at 95°C, a temperature which effectively prevents any non specific amplifications generated in low temperatures. Although it constitutes a state-of-the-art technique for performing hot start PCR the use of reversibly inactive polymerases presents the serious disadvantage of increasing the cost of the DNA amplification reaction significantly, since these enzymes are several times more expensive than the normal ones. For applications that demands the processing of large sample amounts the problem becomes even greater.

[12] Another hot start method is described in Document U.S. PI. No. 5 411 876. In this technique one necessary reactant is physically isolated from the other reactants until a more stringent elevated temperature is reached. The separation between the reactants is achieved by using a small amount of paraffin grease or wax which is placed over a premix containing all but one essential reagent. The paraffin is then melted and sub- sequently cooled down, sealing the premix beneath it. The missing reactant is then placed on top of the paraffin, completely isolated from the other reactants preventing any DNA amplification. When the temperature of the sample surpasses approximately 60⁰C, the paraffin melts, the reagents are merged and the reaction then begins in a more stringent condition. Therefore, the paraffin works as solid barrier that temporarily separates one essential reactant from all the rest.

[13] Although it represents a simple and inexpensive technique to avoid the generation of certain unspecific products, the method has several technical problems, the most critical being the difficulty in retrieving the sample after the reaction has ended and the need for an additional melting step during sample preparation. Also, the temperature at which the merge of reactants occur is around 60⁰C, as opposed to 95°C with the use of more sophisticated hot start methods.

[14] It is clear that the traditional PCR protocol presents limitations and that the methods and products available today for solving the problem of non specific amplifications mainly derived from inter-primer bonding are not entirely satisfying.

[15] It is the purpose of the present invention to provide an efficient, simple, automatic and inexpensive process that can eliminate the problem of primer-dimmer amplification and improve the overall efficiency of the PCR method by a delayed reagent addition after the sample temperature has reached 95°C.

[16] SUMMARY OF THE INVENTION

[17] 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. Although ingenious in principle, 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.

[18] 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.

[19] One of the problems of the original PCR protocol that can be solved by the present invention is the generation of primer-dimmers. In the solution of this problem the invention combines the low-cost of paraffin barrier methods with the efficiency of the use of modified Taq polymerases.

[20] DETAILED DESCRIPTION OF THE PREFERED EMBODIMENTS

[21] In the preferred embodiment, 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. In the preferred embodiment the liquid is an oil, preferably a silicone oil. With the sample heating, in the first PCR cycle, the density of the liquid drops at a higher rate than the density of the aqueous drop containing the separated reactant, which consequently begins to move towards the premix in the bottom of the tube or vessel. The conditions are so that the separated reactant merges with the premix only after the entire sample has been heated to 95°C.

[22] 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.

[23] The efficiency of the present invention is assured by the fact that the reactants are completely separated until the desired moment, or temperature, is achieved. The practicality of the method is given by its automatic nature and by the fact that the material used is liquid at room temperature, which do not creates any difficulty in retrieving the sample after the PCR is done. Finally the low cost of the process is a consequence of its simplicity, of the type of material used, which is preferably a silicone oil, and also of the small volume necessary.

[24] The process of the present invention is additionally explained by means of drawing that illustrates it. Briefly (figure 1), 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. 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. In the preferred embodiment, 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. When the temperature begins to rise, in the first reaction cycle, 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.

[25] The invention is additionally demonstrated by the following example:

[26] EXAMPLE 1

[27] In this example, 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. For this application 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.

[28] In order to assure that the present invention did possess these essential capabilities the physical behavior of the aqueous drop containing the separated reactant was the first aspect to be tested. The correct merging with the premix in the desired moment (after the sample had reached the temperature of 95 degrees Celsius) was visually followed and proven in more than 50 controlled experiments.

[29] Next, the chemical behavior of the aqueous drop inside the oil layer was examined. It was imperative that the oil were insoluble and chemically inert in contact with aqueous solutions and hence could not interfere with the DNA amplification reaction. This oil was insoluble with water in temperatures up to 100 degrees Celsius and its chemical inertia was verified in several DNA amplification reactions in which no affect on amplification yield was seen when using the oil as a simple vapor barrier to prevent sample evaporation (compared with mineral oil, which is often used for this purpose).

[30] Finally, 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. [31] In this example, 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.

[32] Briefly, 200 ul of the silicone oil mixture were poured over 9 ul of an aqueous solution containing all but one necessary PCR reactant. This premix had the following reactant concentrations: 0.5uM of the forward Leishmania chagasi specific primer; 20OuM of each nucleoside triphosphates; 10 mM of Tris-HCl, 5OmM KCl buffer, pH 8.9; 1.5mM MgCl₂; two units of Taq DNA polymerase; and finally, lOpg of purified L. chagasi DNA.

[33] Three sample groups were tested; in the first one, 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.

[34] 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.

[35] 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).

[36] The results of this comparison show the presence of small non specific products

(primer-dimmers) in the samples amplified by the traditional PCR protocol, in which no kind of hot start method is used (lanes 1-3, inferior part of the gel) and the absence of these products in samples amplified by the hot start provided by the process of the present invention (lanes 4-6), as well as by the state-of-the-art hot start method used as the 'gold standard' (lanes 7-9). The results also show that 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).

Claims

[1] A method for impairing the generation of non specific products by the polymerase chain reaction, said method comprising the steps of: a) Placing an aqueous suspension containing all but one or more excluded and essential PCR reactants in an appropriate tube or vessel; b) Covering said aqueous suspension completely with a layer of a dense and chemically inert liquid substance; c) Placing a small aqueous drop containing said one or more necessary reactants inside said dense and chemically inert liquid in a position of complete separation and maximum distance from said suspension containing the other necessary reactants; d) Subsequently performing the DNA amplification reaction.

[2] the method of claim 1 in which said aqueous drop containing one or more necessary reactants is immobilized in its initial position by the density of said liquid layer at room temperature;

[3] the method of claim 1 in which the density of said dense and inert liquid, at room temperature, is equal or approximately equal to the density of said aqueous drop containing one or more necessary reactants;

[4] the method of claim 1 in which said dense and inert liquid layer, at room temperature, works as a physical barrier completely separating said aqueous drop containing one or more necessary reactants from the other reactants at the bottom of the said tube or vessel;

[5] the method of claim 1 wherein the separation effected by said dense and inert substance is automatically revoked by the intrinsic heating of the PCR procedure;

[6] the method of claim 1 in which the density of said dense and inert liquid upon heating becomes smaller or remains smaller then the density of the said aqueous drop containing the necessary separated reactants;

[7] the method of claim 1 in which the separated reactants are automatically merged only in the first DNA denaturation step of the PCR procedure in temperatures above at least 85 degrees Celsius;

[8] the method of claim 2 wherein said dense and inert liquid has a density approximately between 0.9 g/ml and 1.2 g/ml;

[9] the method of claim 2 in which said dense and inert liquid material remains liquid at least in a temperature range between 0 and 100 degrees Celsius;

[10] the method of claim 2 wherein said dense and inert liquid is insoluble in water;

[11] the method of claim 8 wherein said dense and inert liquid is preferably an oil of any origin, as long as it possesses the features and is able to perform the functions described in these claims;

[12] A kit for carrying out the method of claim 1, containing said dense and inert liquid described in the previous claims, the necessary reactants for performing a PCR protocol and any necessary additional component.