GB2324865A - Assay of amplification of a nucleic acid sequence involving conversion of pyrophosphate into inorganic orthophosphate - Google Patents

Abstract

A process for the assay of the efficiency of amplification of a nucleic acid sequence comprises: (i) amplification of said sequence in a reaction mixture comprising at least one dNTP, which results in the generation of pyrophosphate; (ii) addition of an agent, preferably an inorganic pyrophosphatase, which converts said pyrophosphate into inorganic orthophosphate; (iii) detection of said orthophosphate, thereby to determine amplification of said sequence. The reaction mixture may be modified, preferably by the addition of other reagents, in order to reduce the non-specific generation of pyrophosphate and/or inorganic phosphate. The process is of use in the detection and/or diagnosis of microorganisms and viruses from clinical and environmental sources.

Description

Materials and Methods For Detectina Amplified Nucleic Acids Field of the invention.

The present invention relates to materials and methods for detecting amplified nucleic acids following an amplification reaction such as a polymerase chain reaction (PCR). Particularly, but not exclusively; the present invention relates to the detection of the byproducts of such amplification reactions as surrogate markers for successful specific amplification of the target nucleic acid sequences.

Backaround of the invention.

Major developments in DNA and RNA amplification technology have transformed the way DNA and RNA analysis is carried out in both research and clinical laboratories. One such technology, the polymerase chain reaction is a powerful tool for the amplification of trace amounts of nucleic acids and has facilitated the development of a variety of nucleic acid based systems for the development of diagnostics for the detection of viral and bacterial pathogens, genetic disorders, cancer and forensic analysis (for review: Reischl, U & Kochanowski, B. Molecular Biotechnology, 3, 55-71, 1995).

The PCR involves the in vitro enzymatic synthesis of millions of copies of a specific DNA segment through a cyclical reaction based upon the denaturation of the target DNA, annealing of two oligonucleotides primers that flank the target region in DNA and their extension by DNA polymerase. The use of a thermostable DNA polymerase, Taq DNA polymerase, allows simple automated thermal recycling in a single tube. Other methods for amplification based on the same principle but with different reagents have been devised including Q-beta amplification (Lizardi, PM & Kramer, FR. TibTech 9, 5362, 1991).

Patent Nos. 4683195 (Mullis et al 1987) and 4965188 (Mullis et al 1990) described some methods of detecting amplified DNA. These methods as well as others known previously by those in the field commonly include physical separation of the amplified DNA from the other components of the reaction assay by gel electrophoresis, chromatography or capture of the amplified DNA on a solid phase and subsequent detection by immunological methods (Reishl & Kochanowski, Molecular Biotechnology 3, 5571,1995). Detection may be simplified through the use of labels such as radioactivity, enzymes (e.g. alkaline phosphatase), distinct reporter molecules (digoxigenin or biotin) incorporated into the final amplified product using labelled deoxyribonucleotides as substrates. Other methods avoid the separation step and detect the amplified DNA by electrochemical or fluorescence methodologies which rely on the ability of chemiluminescent or fluorescent chemicals such as ruthenium, acridinium or phenanthridium derivatives to intercalate into amplified DNA (Arnold, LJ. et al., Clin. Chem. 35, 1588-1594, 1989: Kenten, JH. et al., Clin. Chem. 37, 1626-1632, 1991: patent No. 5582984, Bienarz, et al, 1994).

All of these methodologies rely on detection of the amplified DNA as a product of the PCR reaction. The PCR reaction can be described as the reaction sequence: Template DNA + dNTPs = amplified DNA +PPi(pyrophosphate).

Detection of PPi as a marker for successful extension of template DNA to provide one additional copy of the template DNA and the use of this to sequence-DNA or detect mutations has been described previously (WO 93/23562: W089/09283: Analytical Biochemistry, 208, 171175, 1993). However the application of the teachings of these papers to detect the successful amplification of DNA by the PCR reaction is unlikely to be suitable since PPi is chemically unstable in aqueous solution and spontaneously hydrolyses to inorganic phosphate (Pi). The thermal recycling process of the PCR reaction accelerates this hydrolysis. One aspect of the invention as disclosed in the applicant's corresponding earlier application (UK 9708892.6) is the conversion of PPi to Pi and the detection of Pi as a measure of successful amplification of template DNA.

EPA 94201768.2 describes the detection of inorganic orthophosphate after PCR amplification and cites an example which shows increased release of Pi during the PCR. The example given also demonstrates the presence of significant levels of Pi in a control sample in which no template DNA was present.

Summarv of the invention.

The present applicant has appreciated that for successful commercial exploitation of methods based upon detection of PPi or Pi, reduction in the non-specific generation of either of these products during the PCR assay is desirable. The major sources of background effects (in addition to actual PPi or Pi contamination of reagents) are (1) the spontaneous hydrolysis of dNTPs to dNMPs and PPi which occurs at an elevated rate on heating; (2) the possible enzymatic conversion of dNTPs to dNMPs and PPi by the exonuclease activities of DNA polymerases; and (3) exonuclease activity cyclically removing dNMPs newly added to the template, which would result in an increased release of PPi without actual amplification of the target DNA sequence. Methods to address these problems would include judicious selection of pure reagents, derivation of dNTP's analogues which will still participate in the PCR process but will not undergo spontaneous hydrolysis, or the addition of inhibitors of hydrolytic or exonuclease activities.

Therefore in accordance with the present invention there is provided a method of determining amplification of a nucleic acid sequence, said sequence having been amplified in a reaction mixture containing one or more dNTPs resulting in the generation of pyrophosphate; comprising the steps of a) adding an agent to the reaction mixture to convert pyrophosphate to inorganic orthophosphate; and b) detecting the presence of said inorganic orthophosphate in the reaction mixture to determine amplification of said nucleic acid.

As a further aspect, there is provided a method of determining amplification of a nucleic acid sequence comprising the steps of a) amplifying said nucleic acid sequence in a reaction mixture containing one or more dNTPs resulting in the generation of pyrophosphate; b) adding an agent to the reaction mixture to convert pyrophosphate to inorganic orthophosphate; and c) detecting the presence of said inorganic orthophosphate in the reaction mixture to determine amplification of said nucleic acid.

In order to minimise or reduce the non-specific generation of pyrophosphate or inorganic orthophosphate, the reaction mixture may be controlled or modified.

Methods of avoiding such non-specific generation would include judicious selection of pure reagents, derivation of dNTP analogues which will still participate in the PCR process but will not undergo spontaneous hydrolysis or the addition of inhibitors of hydrolytic or exonuclease activities. Modification may include the addition of reagents to reduce non-specific generation of pyrophosphate or inorganic orthophosphate. Reagents for the reduction of non-specific generation of pyrophosphate or inorganic orthophosphate may be nucleotide or deoxynucleotide monophosphates and their derivatives, manganese or other transition metal analogues or a halide such as fluoride.

One specific aspect of the invention is treatment of the PCR reaction mix with reagents, e.g. inorganic pyrophosphatase enzyme, to reduce the non-specific levels of Pi and to maximise the conversion of PPi to Pi and thereby provide sufficient discrimination between test and control sample to allow rapid semiquantitative measure of the successful and specific amplification of the target DNA.

A well established assay for Pi is based upon Fiske Subbarow reagent. In this assay the phosphate ions are reacted with ammonium molybdate at acid pH forming ammonium phosphomolybdate. Reduction of this compound produces a blue phosphomolybdenum complex (heteropolymolybdenum). Quantitative levels of Pi can be measured at O.D.660nm or qualitatively by eye. Another aspect of the present invention is the use of assay of Pi based upon ammonium molybdate reagents as the basis of simple methodologies to detect successful performance.

Therefore, the present invention relates to the treatment or modification of the reagents or products of the PCR reaction (or related amplification methods) so as to optimise the conversion of PPi to Pi and reduce the non-specific generation of Pi, to provide an assay based upon the detection of Pi as a semi-quantitative measure of the specific and efficient amplification of target DNA. Such measurement provides a sensitivity equivalent to measurements based upon observation of ethidium bromide stained amplified product following polyacrylamide gel electrophoresis. The present invention offers considerable advantages over the latter and some other methods of detecting amplified products in terms of speed, low cost, use of simple reagents, simple colorimetric detection at visible wavelength without the need for additional instrumentation or laboratory procedures and equipment. The invention is also compatible with instrumentation for high-throughptit automatic analyses.

Aspects and embodiments of the invention will now be illustrated, by way of example, with references to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art.

Brief descriDtion of the Figures.

Fig. 1 illustrates the measurement of increasing concentrations of Pi during successive PCR cycles and correlation with increasing formation of amplified DNA as measured by ethidium bromide staining of amplified DNA after polyacrylamide gel electrophoresis; Fig. 2 compares the detection of amplified DNA and Pi when different components of the PCR assay are omitted and demonstrates that there is a significant level of non- specific release of Pi; Fig. 3 illustrates that through the use of pyrophosphatase enzyme the level of Pi detected is proportional to the amount of starting DNA template; and Fig. 4 illustrates that treatment with higher concentrations of pyrophosphatase reduces the nonspecific level of Pi detected.

Detailed descriPtion.

EXAMPLE 1.

Vector pPD8, an expression vector for single chain antibody fragments was used as source of template DNA.

The vector contains a gene sequence encoding the variable light chain of anti-atrazine antibody (Byrne,F.et al., Food & Agricultural Immunology, 8, 19-29,1996).

Oligonucleotide primers VK1-Back (5' GACATTGAGCTCACCCAG TCTCCA-3') and VK-Forward (5' GTTAGATCTCGAGCTTGGCCC-3') amplify anneal to the 5' and 3' ends respectively of the variable light chain gene.

For each PCR assay of total volume 50ul, 25pmoles of each primer was mixed with 0.lng of template in a reaction mixture containing 2mM MgCl2, 16mM (NH4)2SO4, 67mM Tris-HCl, pH 8.0, 0.01% Tween 20, 0.Spmoles of each of dATP, dGTP, dCTP, dTTP. Tubes were placed at 940C for Smin and then 0.025units taq-polymerase added. Tubes were then subjected to repeated thermocycling under conditions of 940C for 1 min, 500C for lmin, 720C for 1 min.

At the end of the appropriate number of cycles, Sul of reaction mix was removed from each tube and electrophoresed through 1% agarose gel containing ethidium bromide and photographed under W illumination.

The remaining 45ul of each assay was used to measure the amount of Pi (inorganic phosphate) by adding sequentially 400wul of 10% trichloroacetic acid, 600ul of H2O, 200ul acid molybdate solution (Sigma Chemical Company, UK, cat no. 661-1) and 50ul of Fiske & SubbaRow solution ( Sigma Chemical Co, UK. Cat No. 670-A). After 10min, absorption at 660nm was measured.

The results shown in Fig. la, indicate that amplified DNA product is visible by ethidium bromide staining after 20 cycles of PCR. From the results in Figure 1b it can be seen that there is a successive rise in the level of inorganic phosphate with increase PCR cycles. After 20 cycles, at which time a band was visible in Fig. la, a reading of 0.77 is recorded.

Absorption readings in excess of 0.5 are visible with the naked eye as a blue colour. Successful performance of the PCR reaction can then be visualised by eye after 20cycles.

EXAMPLE 2 The PCR reaction was carried out as described in Example 1 but with individual components of the PCR assay not present.

Fig. 2 compares the detection of amplified DNA and Pi when different components of the PCR assay are omitted. It can be seen that while the level of Pi is highest when successful DNA amplification occurs, there is a significant level of non-specific release of Pi. In this example, the presence of an apparent trace of template contamination in tube 1 is also reflected in a slight increase in Pi released. That the background Pi release is due to the spontaneous hydrolysis of dNTPs was confirmed by the release of Pi by subjecting dNTPs alone to cyclical heat cooling (results not presented).

Alterations of PCR assay conditions through different heat/cooling regimes or assay buffer pH could modify but not eliminate this non-specific release of Pi.

EXAMPLE 3 After completion of the PCR reaction as described in Example 1, 1 unit of inorganic pyrophosphatase (Sigma Chemical Co, UK) was added to each 45ul assay and incubated at room temperature for l5min prior to detection of Pi. Fig. 3 shows that the level of Pi detected is proportional to the amount of starting DNA template and may therefore be used to quantitate the amount of target DNA within a sample. It was also found that higher concentrations of enzyme (10 units) reduced the non-specific signal (Fig. 4).

Claims (6)

1. A method of determining amplification of a nucleic acid sequence, said sequence having been amplified in a reaction mixture containing one or more dNTPs resulting in the generation of pyrophosphate; comprising the steps of a) adding an agent to the reaction mixture to convert pyrophosphate to inorganic orthophosphate; and b) detecting the presence of said inorganic orthophosphate in the reaction mixture to determine amplification of said nucleic acid.

2. A method of determining amplification of a nucleic acid sequence comprising the steps of a) amplifying said nucleic acid sequence in a reaction mixture containing one or more dNTPs resulting in the generation of pyrophosphate; b) adding an agent to the reaction mixture to convert pyrophosphate to inorganic orthophosphate; and c) detecting the presence of said inorganic orthophosphate in the reaction mixture to determine amplification of said nucleic acid.

3. A method according to claim 2 wherein the reaction mixture is modified to reduce non-specific generation of pyrophosphate or inorganic orthophosphate.

4. A method according to claim 2 or claim 3 wherein additional reagents are added to the reaction mixture to reduce non-specific generation of pyrophosphate or inorganic phosphate.

5. A method according to any one of the preceding claims wherein the agent is inorganic pyrophosphatase.

6. A method according to any one of the preceding claims wherein the amplification of the nucleic acid sequence is used for the detection and/or diagnosis of microorganisms and viruses from clinical or environmental sources.