WO2003048393A1

WO2003048393A1 - Mixture of two partially denaturating agents for the preparation of pcr buffer solutions

Info

Publication number: WO2003048393A1
Application number: PCT/IT2001/000611
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: AU2002217428A1

Abstract

The object of the invention is a new mixture of two partially denaturating agents to be used for the preparation of PCR buffer solutions in order to enhance the rate of the DNA denaturation reactions.

Description

MIXTURE OF TWO PARTIALLY DENATURATING AGENTS FOR THE PREPARATION OF PCR BUFFER SOLUTIONS

The invention concerns DNA amplification techniques and more particularly the use, in buffer solutions for the polymerase chain reaction (PCR) , of a new mixture of two partially 5 denaturating agents: glycerol and dimetyl sulfoxide (DMSO) . These two agents are able to facilitate DNA denaturation and to minimise, at the same time, the disadvantages related to the use of denaturating agents in PCR buffer 10 solutions.

As already known, PCR (polymerase chain reaction) is surely the technique that has completely changed the molecular biology permitting the fast, cheap and efficient

15 synthesis of DNA.

The PCR process comprises three subsequent steps, which are repeated at every single denaturation cycle:

1. During the first reaction (denaturation) the 20 double elix of DNA opens to the two complementary single DNA strands.

2. In the second step, called annealing reaction, short sequences of single DNA strand of 20 bais pairs, called primers, because of DNA

25 complementarity, bound the sequences adjacent to each end of the called target region. 3. In the last step, called polymerisation reaction, DNA polymerase (the enzyme responsible for DNA replication) synthesised from the 3' -OH end a single DNA strand that is complementary to the sequence to be amplified.

The first denaturation step is surely the main reaction and without it the amplification cycle can not proceed. During this reaction, the double elix of DNA loses its primary structure due to hydrogen bounds between nucleotides and hydrophobic interactions between stacked basis and it loses the secondary structures due to close complementary sequences which, within the same molecule, form loops or crosses. In the common laboratory practice, DNA denaturation is induced by temperature increasing, by using chemical agents which destabilised the double elix structure or by using extreme pH conditions that gives to nucleotides a net charge and enhance denaturation .

The best known and common denaturating agents are formamides, non ionic detergents and dimetylsulfoxide (DMSO) . These compounds have many disadvantages, for example: formamides damages polymerase physical -chemical characteristics; non ionic detergents enhance aspecific amplification rate.

DMSO causes DNA breaking and polymerase inactivation, on the other hand, it drastically reduces the formation of secondary structures that stop polymerase progression during replication, and furthermore it can induce CG- rich regions denaturation and can improve annealing specificity of the primer-template complex creating more strict reaction conditions, ensuring the bound between each primer and the corresponding DNA sequence .

Nowadays, commercially available buffer solutions, contain DMSO in a rate higher than 3%, usually between 3% and 10%, and do not contains compounds capable to minimise DMSO negative effects on DNA sequence preventing and on polymerase activity.

The present invention overcomes the problems related to the use of denaturating agents, like DMSO, in the preparation of PCR buffer solution, providing a new mixture for the preparation of PCR buffer solutions able to reduce DMSO rate and containing compounds enabling the denaturation to be executed at a lower temperature, it can stabilises DNA polymerase and it reduces the risk of incorrect annealing.

This is obtained using, according to the present invention, a new mixture of DMSO and Glycerol where each component shows the following features : Glycerol : a) It's a co-solvent that allows the denaturation reaction to be carried out at lower temperatures. b) It stabilises DNA polimerase c) It reduces false-priming that occurs when primers bound to sequence different from the one to be amplified. This characteristic is very important in multiplex PCR when a large number of primers must bound each to a precise template sequence. DMSO: a) It helps DNA denaturation especially of GC- rich regions; b) It improves specificity pairing between primers and templates.

At the same time, experimental results, have underlined that the two components of the mixtures show, out of their own characteristics, an high synergic activity, which gives to the containing buffer solutions the unique features as follows:

DMSO in low concentrations, thanks to the combination with glycerol, does not damage DNA polymerase (which is quite expensive) enabling the use of lower amounts of the enzyme; it offers an high rate of amplification trough regions of the DNA molecule having different danaturation profiles, and it renders the entire process cheaper; it further avoids the formation of intrachain bounds that stop polymerase progression during replication; it permits the use of a concentration of DMSO which is lower than the one commonly used in buffer solutions in the state of the art, avoiding damages of the DNA molecule to be amplified and the risk of enzymes inactivation; it reduces dangerous false-priming between primers and templates, useful particularly in long PCR when the sequences to be amplified have a length up to 18.000 bais pairs; it allows to have a good DNA polymerase processivity even after long periods of exposure to high temperatures and/or the same denaturating agents .

Figure 1 compares the results obtained from a PCR in presence of a buffer solution containing only glycerol at different concentration with those obtained from a PCR in presence of a buffer solution containing glycerol in a concentration of 1% together with DMSO at different concentrations. Axis of ordinates represent band intensity of an electrophoresis test of amplified samples, the minimum amplified quantity is normalised at value 1.

Data represented in the graphic are listed in the following table:

% glycerol glycerol glycerol + 1% DMSO

0% 2,5 3,5

3% 3 5

6% 7 9 8% 7 11

10% 7 8,5

12% 3 4

15% 1 1

The mixture, object of the present invention is characterised in containing a very low amount of DMSO.

The mixture, in the final concentration used for the amplification reaction is as follows: DMSO from 0.8 to 1.5 % GLYCEROL from 7 to 9 % In a preferred embodiment the mixture, object of the present invention, is characterised in having the following composition in the final concentration used for the amplification reaction:

DMSO 1 %

GLYCEROL 8 % In conclusion, the use of the new mixture of DMSO and glycerol, for the preparation of PCR buffer solutions allows the optimisation of the amplification reaction rate because the combination between glycerol and DMSO enables the use of amounts of DMSO lower than those known up to now, and this fact consequently reduces all the inhibiting effects on polymerase and the risks of damaging the DNA molecules to be syntetised. PCR buffer solutions containing the mixture of partially denaturating agents, object of the present invention, are extremely suitable for GC- rich regions PCR, for multiplex PCR and for Long PCR.

Claims

1. Use of DMSO and glycerol for the preparation of PCR buffer solutions.

2. PCR buffer solution characterised in that, it comprises the following components in a final concentration used in the amplification reaction: DMSO from 0.8 to 1.5 %

GLYCEROL from 7 to 9 %

3. PCR buffer solution according to claim 2 characterised in that DMSO is an a concentration of 1% and glycerol is in a concentration of 8%.