WO2003048392A1 - Salts mixture for the preparation of pcr buffer solutions - Google Patents

Abstract

The invention relates to the use of a new salts mixture to be used for the preparation of PCR buffer solutions in order to offer high amplification uniformity at different temperatures and an higher reaction efficiency with different DNA polymerases.

Description

SALTS MIXTURE FOR THE PREPARATION OF PCR BUFFER SOLUTIONS

The present invention relates to DNA amplification techniques and more particularly to the use of Potassium glutamate and Ammonium Acetate in PCR buffer solutions in order to offer high amplification uniformity at different temperatures and higher reaction efficiency with different DNA polymerase or cocktails of two enzymes having different characteristics. As known, PCR (polymerase chain reaction) is surely the technique that changed completely molecular biology allowing inexpensive, fast and efficient DNA synthesis.

The PCR reaction is based on the repetition of a single amplification cycle made of three subsequent steps :

1. During the first reaction (denaturation) the double elix of DNA opens to the two complementary single DNA strains. 2. In the second step, called annealing or hybridisation, short single strand DNA sequences of 20 bais pairs, called primers, because of bais complementarity, bound to the regions adjacent to each end of the sequence to be amplified, called target sequence.

3. During the last step, DNA polymerisation take place and DNA polymerase (the enzyme responsible for replication) syntetises, from the primers 3'- OH end, a single strand of DNA complementary to the desired sequence to be syntetised. In the laboratory practice, the choice of the PCR buffer is the most critical moment for the success of experimental protocols because the proceeding of the three reactions of the basic PCR amplification cycle depends on the physical and chemical characteristics of the reaction environment, which is mostly defined by the same buffer solution.

Moreover, to render the entire process economically favourable, the use of efficient buffer solutions for various amplification protocols for the analysis of different types of DNA samples with various polymerase or temperature intervals, is desiderable.

One of the most frequent problem related to the preparation of PCR buffer solutions is the achievement, in the buffer solution, of a suitable ions concentration in order to obtain homogeneous denaturation and high specificity of the hybridisation reaction between primers and templates sequence. In fact, it is well known that cations concentration affect DNA denaturation temperature and different anions act differently on DNA sequences having different structural features. Another problem is related to the use of different DNA polymerases or cocktails of two different polymerases, as in Long PCR. In this case, the buffer solution should ensure the maximum efficiency for all the enzymes. At the present time, PCR buffer solutions capable of overcoming all the above described problems are not commercially available.

The present invention overcomes said disadvantages providing the use of a mixture of Potassium Glutamate and Ammonium acetate for the preparation of PCR buffer solutions. Said mixture is unknown in the state of the art and among commercially available products. The buffer solution comprises the salt mixture of Potassium glutamate and Ammonium Acetate and, according to the present invention, shows the following advantages:

It ensures high amplification uniformity at various temperatures because the salt mixture is capable of rendering the local DNA elix denaturation point homogeneous even if sequences having dishomogeneous denaturation profiles are present inside the same molecule; it enables an high specific primer-template hybridisation; it permits the use of primers mixtures having various melting points; it offers high efficiency in presence of different types of polymerases, namely different enzymes, or cocktails of more than one enzyme having different characteristics.

The high efficiency outcome from the fact that those anions are plentifully present in bacteria and their use permits to restore enzymes natural working conditions, giving them the higher processivity and functional stability; it helps DNA denaturation even of very long sequences (up to 18.000 bp) because in presence of the two salt the DNA denatures easily; it avoids, during annealing, hybridisation within sequences inside the same DNA molecule

(intrachain annealing) ; it allows the treatment of amplified DNA samples, obtained from PCR, with restriction or modification enzymes without any preliminary washing step or without needing to change the buffer solution because the salts do not interfere with enzymes catalytic activity.

According to the present invention a PCR buffer solution comprising a mixture of Potassium Glutamate and Ammonium Acetate is provided, in order to obtain the following final concentration, to be used in the amplification reaction: POTASSIUM GLUTAMMATE from 1 to 250 mM AMMONIUM ACETATE from 5 to 200 mM

In a preferred embodiment the mixture object of the present invention is characterised in having the following final concentration to be used in the amplification reaction:

POTASSIUM GLUTAMMATE 20 mM

AMMONIUM ACETATE 60 mM

In conclusion, the use of the mixture of the two salts Potassium Glutamate and Ammonium Acetate allows the preparation of PCR buffer solutions particularly suitable for the following amplification protocols:

Multiplex PCR since it enables high amplification uniformity at different temperatures, permitting the utilisation of mixtures of primers having various melting points and it ensures great hybridisation specificity between primers and templates;

Long PCR since it allows high efficiency in the presence of different types of polymerases, i.e. with various enzymes or cocktails of many enzymes with different features, it facilitates DNA denaturation even in case of very long DNA sequences (up to 18.000 bp.) .

Claims

CLAIMS 1. The use of a mixture of Potassium Glutamate and Ammonium Acetate for the preparation of PCR buffer solutions.

2. PCR buffer solution characterised in that Potassium Glutamate and Ammonium Acetate in their final concentration used in the amplification reaction are present in the following rate:

POTASSIUM GLUTAMMATE from 1 to 250 mM AMMONIUM ACETATE from 5 to 200 mM

3. PCR buffer solution as in claim 2 characterised in that Potassium Glutamate has a concentration of 20 mM and Ammonium Acetate has a concentration of a 60mM.

4. PCR buffer solutions as in claim 1 and 2 characterised by the fact of being used in multiplex PCR.

5. PCR buffer solutions as in claim 1 and 2 characterised by the fact of being used in Long PCR .