WO2003048391A1

WO2003048391A1 - Double buffer system for polymerase chain reaction buffers

Info

Publication number: WO2003048391A1
Application number: PCT/IT2001/000609
Authority: WO
Inventors: Vincenzo Nigro
Original assignee: Bio3 Research Srl
Priority date: 2001-12-04
Filing date: 2001-12-04
Publication date: 2003-06-12
Also published as: AU2002217426A1

Abstract

The invention relates to buffer solution characterised in that they comprise a double buffer system Tris-Hepes to be used, alone or together with Potassium Glutamate to reach the desired pH level without adding Chloridric Acid or Potassium Iodide. Several preferred embodiments to be used for many different experimental protocols for the DNA analysis of DNA having different chemical-physical characteristics or coming from heterogeneous sources are described.

Description

DOUBLE BUFFER SYSTEM FOR POLYMERASE CHAIN REACTION BUFFERS.

The present invention relates to the field of molecular biology and more particularly concerns the use, in buffer solutions for DNA amplification techniques, of a double buffer system Tris-Hepes enabling to reach the desired pH value without adding Cloridric Acid or Potassium Iodide, offering maximum stability during temperature variations, maximum stability of the used enzymes, even after a cyclic or extended exposure to high temperature, and maximum amplification uniformity at various temperatures. Said double buffer system is used in a reaction mixture also comprising salts that allows the interactions between DNA helix. Usually, in most of the buffer solutions, those functions are carried out by Potassium Chloride. In the present preparation, the specific use of Potassium Glutamate is mentioned because it is universally present in the bacterial environment. The polymerase chain reaction (PCR) is nowadays the faster and most reliable technique for DNA sequences amplification. This method, that has been described for the first time in the US patent No. 4,683,195 and in the US patent No. 4,683,202, changed completely molecular biology because it turned out DNA extraction, amplification and analysis extremely rapid and inexpensive. PCR is based on the basic concept which states that if the two sequences placed close to each end of the DNA fragment, that has to be amplified (called target region) , are known, it is possible to replicate said fragment many times obtaining in a short period of time an enormous number of copies of the same.

PCR consists in the sequential repetition of a single amplification cycle, which comprises tree subsequent steps :

The first step is the denaturation reaction: DNA sample undergoes to rapid heating at a temperature around 95° C for approximately one minute. In this way, the DNA molecule loses its secondary structure and the double helix (DNADds) denaturates in two single complementary strains (DNAss) . Each single strain works as a template for the synthesis of an other complementary DNA strain. In the second step, called annealing or hybridisation, the temperature is lowered around 50-60 °C, and short single strain DNA sequences of 20 bais pair, (called primers) , are added. Those short sequences are absolutely necessary for the reaction prosecution because DNA polymerase can, walking on the template sequence, add nucleotides only if a free 3 'OH end is present. The primers, having a known or a random nucleotide sequence (random primers) bound to the regions adjacent to the target sequence only if they show a high level of complementarity.

During the last step, DNA polymerase (the enzyme responsible for DNA replication) is added, at an optimal temperature of 68-72 °C and this allows the synthesis, starting from the primers 3' -OH end, of a single DNA strain complementary to the target sequence. At this temperature, primers that are not complementary to the target region are not able to stay bound and do not induce the DNA fragment extension; this phenomenon is responsible for the reaction sequence specificity. The entire cycle is repeated for 20-40 times starting again the process with the denaturation step. In theory, the number of copies obtained raises exponentially, doubling at every cycle, until a plateau is reached where the collected amount of DNA to be amplified is higher than the amount the enzyme can amplify in a single cycle. Thermophylic bacterial polymerases are the most commonly used enzymes because they are stable and active after the repeated heating steps of the denaturation-annealing cycle. In this manner, the target region can be amplified up to 4 x 10^ times after 25 cycles.

The use of specific buffer solutions enables reaction yield and specificity optimisation as well as the achievement of a suitable reaction environment for the various primer-template combinations and different application of said method. Said buffer solutions can comprise compounds that, during the first step, facilitate the denaturation of CG-rich regions and inhibit secondary structures formation; during the second step, eliminate aspecific primer-DNA bounding and stabilise the bounds within the primer-template complex; even more, during the last step, they activate and stabilise DNA polymerase.

The main problem related to the preparation of PCR buffer solutions is ensuring enzyme activity preservation, maintaining its characteristic during continuous temperature sudden changes, and guaranteeing pH uniformity during repeated reaction cycles. Moreover, obtaining buffer solution suitable for different

PCR technique application, like the analysis of various types of DNA molecules, for the contemporary amplification of many sequences

(multiplex PCR) , for the amplification of long DNA sequences (Long PCR) , the amplification of sequences having an high rate of bais homology (A/T-rich or G/C-rich) , analysis of mutant sequences, analysis of fragmented DNA samples, or limited amount of template DNA as viral DNA is desiderable. Finally, obtaining buffer solutions that do not damage automatic DNA analysis instruments, for example High efficiency DNA chromatography systems (HPLC and DHPLC) is also desiderable . Nowadays, using the double buffer system

TRIS-HC1, a pH decreasing of 0,3 every 10 °C temperature increasing is detected. As a matter of fact, this system, like many other commonly used buffers, cannot control sudden pH decreasing at the denaturation temperature. This pH decreasing causes the depletion of purines (lost of Adenine and Guanine) with the creation of abasic sites, and induces DNA template helix breaking, which cannot be trancripted anymore.

This phenomenon is directly proportional to the length of PCR reaction products and inversely proportional to the initial DNA template quantity. The invention task is the overcoming of said disadvantage, providing a double buffer system

Tris-Hepes (base-acid) , unknown in the state of the art, which allows the maintenance of a steady pH during PCR steps, and shows the following further advantages : a) In case of PCR multiplex, when many different DNA fragments must be amplified at the same time, a steady pH at various temperatures allows to obtain a higher reaction yield. b) All the used enzymes (DNA polimerase) show high stability even after a cyclic and extended exposure to high denaturation temperature, because they are well buffered. The double buffer system, object of the invention, each component are provided in the following amount for their final use:

TRIS from 1 to 200 mM HEPES from 1 to 200 mM

In the double buffer system, object of the invention, the components of the double buffer system Tris-Hepes are in a rate between 8:1 and 1:4; and the best rate is between 4:1 and 1:1. The choice of the best value depends on the characteristic that must be given to the obtained buffer solution. In particular, the higher enzyme processivity is detected in the presence of a buffer solution containing Tris-Hepes in a rate of 35:10 and 25:10. In the buffer solution having a rate value of 20:10, and consequently a lower pH value, a low replication error probability is detected, that probably is due to the loss of enzyme processivity. Moreover, the invention advantageously provides the double buffer system Tris-Hepes in combination with Potassium Glutamate (that can be replaced with Potassium chloride KCl) for the preparation of buffer solutions. Potassium Glutamate facilitates the use of bacterial enzymes, because usually those enzyme works in the presence of said salt, and allows the use of various DNA polymerase and/or restriction enzymes for post-PCR analysis without needing salt changes in the reaction buffer. Therefore, the buffer solution composition, used in the amplification reaction is as follow:

TRIS from 1 to 200 mM

HEPES from 1 to 200 mM POTASSIUM GLUTAMATE from 1 to 250 mM

It has to be underlined that the use of Potassium Glutamate (that can be replaced by Potassium chloride KCl) for the preparation of PCR buffer solutions is unknown in the state of the art, and its application to PCR is drawn from the fact that said salt is the most abundant in bacteria and its use in buffer solutions enables, as well as possible, the restoration of enzyme natural functional conditions and guarantees its maximum efficiency.

Preferred embodiments characterised in that the base composition, as described above, is used in combination with other compounds like Magnesium Sulphate, Ammonium Sulphate, Ammonium Acetate, Glycerol, Dimetylsulfoxide (DMSO) , Dithiothreitol (DTT) and gelatin, that permit to obtain specific buffer solutions suitable for different PCR protocols, are shown as follows.

EXAMPLE 1

Magnesium Chloride, Dithiothreitol, (DTT) and gelatin, are added to the double buffer system Tris-Hepes, in association with Potassium Glutamate (that can be replaced with Potassium chloride KCl) , for the preparation of PCR buffer solutions. In the form of an aqueous solution the buffer has the following composition in the final concentration used in the amplification reaction. TRIS from 1 to 200 mM HEPES from 1 to 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM MAGNESIUM CHLORIDE from 4 to 20 mM DTT from 0,01 to 2 mM

GELATIN from 0,001 to 1 % Magnesium Chloride is used because it causes the increasing of deoxyribonucleotides triphosphate (dNTP) which catalyses, together with Magnesium ion Mg²⁺, nucleotides polymerisation on the template, thus guaranteeing a higher polymerase catalytic activity especially useful in multiplex PCR.

Therefore, said buffer solution appears to be particularly good for conventional or quantitative multiplex PCR, conventional PCR of AT-rich sequences and quantitative PCR.

Furthermore, the buffer solution of this example, gives considerable advantages in multiplex PCR because it allows simultaneously high specific amplification of several DNA fragments, as shown in the attached figure 1.

Figure 1 shows the results of a quantitative multiplex PCR on DNA samples containing 10 sequences having a medium length of 190-500 bais pairs in the presence of the buffer solution of the above example or in the presence of other commercially available buffer solutions . The best experimental results were obtained using a buffer solution having the following composition in the final concentration used in the ampli ication reaction:

TRIS 10 mM

HEPES 10 mM

POTASSIUM GLUTAMATE 50 mM

MAGNESIUM CHLORIDE 7 , 5 mM DTT 0,2 mM

GELATIN 0,01 %

Said buffer solution is prepared in the form of optimal mother solution that is five times more concentrated than the final buffer solution used in the amplification reaction.

EXAMPLE 2 Magnesium Sulphate, Ammonium Acetate, DMSO, Glycerol, DTT and gelatin are added to the double buffer system Tris-Hepes, in association with Potassium Glutamate (that can be replaced with Potassium chloride KCl) , for the preparation of PCR buffer solutions. In the form of an aqueous solution the buffer has the following composition in the final concentration used in the amplification reaction.

TRIS from 1 to 200 mM

HEPES from 1 to 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM MAGNESIUM SULPHATE from 1 to 20 mM AMMONIUM ACETATE from 5 to 200 mM

DMSO from 0, 05 to 10 %

GLYCEROL from 1 to 12 %

DTT from 0,01 to 2 mM

GELATIN from 0, 001 to 1 %

The used concentration of Magnesium Sulphate is higher than in other known buffer solutions. This salt causes the increasing of deoxyribonucleotides triphosphate (dNTP) which catalyses, together with Magnesium ion Mg²⁺, nucleotides polymerisation on the template, thus ensuring a higher polymerase catalytic activity especially useful in multiplex PCR and in Long PCR.

The buffer solution of the present example allows to lead the annealing (or hybridisation) reaction in no strict temperature conditions ensuring a high level of reproducibility in several different experimental protocols.

In the laboratory practice, said buffer is suitable for the amplification of small amounts of DNA, for example viral DNA, the amplification of long DNA fragments, conventional and quantitative multiplex PCR and quantitative PCR.

Moreover, the buffer solution of this example gives considerable advantages because it allows to maintain reaction efficiency and specificity during considerable changing of primers-template annealing temperature, ensuring the success of the amplification reaction even in case of electronic or human mistakes, as shown in the following table; in the table the results of a PCR are shown, at different annealing temperatures, using commercial buffer and the buffer as described in the above example.

TABLE 1

As shown in the above table, in the presence of example 2 buffer, the amplification is always specific even when the annealing temperature varies extensively.

Said buffer solution is efficient in the amplification of very long sequences (up to 12kb) , as shown in the following table. TABLE 2

Table 2 shows the results of a Long PCR of a 120 Kilobasis DNA fragment between exon 70 and exon 74 of Distrofina gene on chromosome Xp21, in the presence of commercially available enzymes together with commercial available buffers or with the example 2 buffer. The results are shown as rate "Signal-Rumour" where "Signal" is the amplification of the desired length band, "Rumour" is the aspecific amplification and the sign minus (-) means that in standard conditions PCR did not give any amplification. Moreover, said solution, enables a high stability after a long cyclic exposure to the denaturation temperature, as shown in the following table. Table 3

Table 3 shows the results obtained with the exposure of different commercially available enzymes, to a temperature of 96 °C , for zero, thirty and ninety minutes in the presence of commercial buffer solutions or the example 2 buffer before carrying out a control PCR following a 24 cycles standard protocol. The results obtained with each enzyme are normalised in comparison with the results obtained from the standard amplification in the presence of the commercial buffer and without exposure at a temperature of 96 °C.

The best experimental results were obtained using a buffer solution having the following composition in the final concentration used in the amplification reaction:

TRIS 30 mM

HEPES 10 mM POTASSIUM GLUTAMATE 20 mM

MAGNESIUM SULPHATE 5 mM

AMMONIUM ACETATE 60 mM

DMSO 1 %

GLYCEROL 8 % DTT 0,2 mM

GELATIN 0,01 %

Said buffer solution is prepared in the form of optimal mother solution that is five times more concentrated than the final used buffer solution.

EXAMPLE 3 Magnesium Sulphate and Ammonium Sulphate are added to the double buffer system Tris-Hepes, in association with Potassium Glutamate (that can be replaced with Potassium chloride KCl) , for the preparation of PCR buffer solutions. In the form of an aqueous solution the buffer has the following composition in the final concentration used in the amplification reaction.

TRIS from 1 to 200 mM

HEPES from 1 to 200 mM POTASSIUM GLUTAMATE from 1 to 250 mM

MAGNESIUM SULPHATE from 1 to 20 mM

AMMONIUM SULPHATE from 1 to 70 mM

This buffer is particularly suitable for multiplex PCR, for Long PCR, for mutant sequences analysis, for fragmented samples analysis or samples containing limited amount of DNA like viral DNA. This buffer do not damage automatic

DNA analysis instruments, like DNA liquid phase chromatography (HPLC e DHPLC) , because the buffer solution residues do not interact with the chromatographic phase and do not settle on the chromatographic columns. Moreover, said buffer solution is advantageous because it shows rapid activation capacity when specific DNA polymerases, called "Hot Start" are used. TABLE 4

In table 4 the results obtained from a PCR with "Hot Start" enzymes and in the presence of commercial buffers or example 3 buffer are shown.

TRIS 10 mM

HEPES 20 mM

POTASSIUM GLUTAMATE 10 mM

MAGNESIUM SULPHATE 3 mM

AMMONIUM SULPHATE 10 mM

Claims

1. The use of a double buffer system Tris-Hepes for the preparation of PCR buffer solutions.

2. The use of a double buffer system Tris-Hepes according to claim 1 in association with Potassium Glutamate for the preparation of PCR buffer solutions.

3. PCR buffer solution wherein the following components are present in the final concentration used in the amplification reaction: TRIS from 1 to 200 mM

HEPES from 1 to 200 mM

4. PCR buffer solution according to claim 3 wherein the following components are present in the final concentration used in the amplification reaction:

TRIS from 1 to 200 mM

HEPES from 1 to 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM

5. PCR buffer solution according to claim 3 and 4 having the following composition in the final concentration used in the amplification reaction:

TRIS from 1 to 200 mM

HEPES from 1 to 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM MAGNESIUM CHLORIDE from 4 to 20 mM

DTT from 0,01 to 2 mM

GELATIN from 0,001 to 1 %

6. PCR buffer solution according to claim 3 and 4 having the following composition in the final concentration used in the amplification reaction: TRIS da 1 a 200 mM

HEPES da 1 a 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM MAGNESIUM SULPHATE from 1 to 20 mM AMMONIUM ACETATE from 5 to 200 mM DMSO from 0,05 to 10 %

GLYCEROL from 1 to 12 %

DTT from 0, 01 to 2 mM

GELATIN from 0,001 to 1 %

7. PCR buffer solution according to claim 3 and 4 having the following composition in the final concentration used in the amplification reaction: TRIS from 1 to 200 mM

HEPES from 1 to 200 mM

POTASSIUM GLUTAMATE from 1 to 250 mM MAGNESIUM SULPHATE from 1 to 20 mM AMMONIUM SULPHATE from 1 to 70 mM

8. PCR buffer solution according to claim 5 having the following composition in the final concentration used in the amplification reaction: TRIS 10 mM

HEPES 10 mM

POTASSIUM GLUTAMATE 50 mM

MAGNESIUM CHLORIDE 7 , 5 mM

DTT 0,2 mM GELATIN 0,01 %

9. PCR buffer solution according to claim 6 having the following composition in the final concentration used in the amplification reaction:

TRIS 30 mM HEPES 10 mM

POTASSIUM GLUTAMATE 20 mM

MAGNESIUM SULPHATE 5 mM

AMMONIUM ACETATE 60 mM

DMSO 1 % GLYCEROL 8 %

DTT 0 , 2 mM

GELATIN 0,01 %

10. PCR buffer solution according to claim 7 having the following composition in the final concentration used in the amplification reaction:

TRIS 10 mM

HEPES 20 mM

POTASSIUM GLUTAMATE 10 mM

MAGNESIUM SULPHATE 3 mM AMMONIUM SULPHATE 10 mM

11. PCR buffer solution according to claims 2, 4, 5, 6, 7, 8 ,9 ,10 characterised in that Potassium Glutamate is replaced by Potassium Chloride the same concentrations.