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Analytical method and kit

Analytical method and kit description/claims

The present invention relates to a method of analysing a nucleic acid sequence, for example to detect the presence of a particular sequence within a sample, or to determine the precise sequence of a particular nucleic acid, and to kits and reagents for use in these methods.

Currently there is a wide range of methods of conducting analysis of nucleic acids. Analytical methods may be directed to those that detect the presence or amount of a particular nucleic sequence in a sample suspected of containing that sequence. Other methods elucidate the structure of a nucleic acid to determine its sequence of nucleotides for information or diagnostic purposes.

Amplification reactions are commonly used to effect or assist in this analysis, particularly where the particular nucleic acid sequence is present in only minute amounts. The use of amplification reactions such as the polymerase chain reaction (PCR) for detection of target nucleic acid sequences is well known. One or more primers which are specific for the particular sequence are included in an amplification reaction mixture. These will hybridise to the specific target sequence when in single stranded form within a sample tube. If the target sequence is present, the primer will bind to it, whereupon polymerase enzyme present in the mixture will, at certain temperature conditions, extend the primer to form a complete complementary strand. This material then forms further templates, for amplification in subsequent cycles of denaturation, primer annealing and extension.

The amplified product may be detected, for example on an electrophoretic gel. However, fluorescent labelling methods are now frequently used to detect when an amplification reaction has been effected, and/or to monitor its progress. Examples of such assays include the TAQMAN™ assay, as well as assays described and claimed for example in WO 99/28500, WO 99/28501, WO 99/42611 and WO 99/66071. An assay using labelled ribo-oligonucleotide probes is described in WO 98/04738. Labelling of probes however is a complex process which increases the cost.

Methods for sequencing nucleic acid sequences are also well known. Gel methods are conventional. More recent methods are carried out using devices such as the Pyrosequencer available from Pyrosequencing AB, rely on the generation of a visible signal when a correct nucleotide is added during the construction of a complementary strand on a single stranded nucleic acid template. Other methods for interrogating the identity of a specific base in a nucleic acid sample using pyrophosphorolysis reactions are described in WO 99/46409.

The applicants have found that RNA probes, which are unlabelled, can provide an advantageous means for monitoring or detecting such events.

According to the present invention there is provided a method for detecting or analysing a nucleic acid sequence in a sample, said method comprising contacting said sequence with an RNA probe under conditions such that the probe will bind to the sequence, subjecting any nucleic acid/probe complex to conditions under which RNA probe bound to nucleic acid is hydrolysed to generate adenosine monophosphate (AMP), detecting AMP produced, and relating this to the presence or nature of the nucleic acid sequence in the sample.

RNA probes may be readily hydrolysed by a variety of enzymes, when in double stranded form. These include polymerase enzymes commonly used in PCR reactions such as Taq polymerase. Alternatively they may be hydrolysed by RNAse enzymes, which will hydrolyse them only when in double stranded form, for example as an RNA/DNA duplex. Such duplexes may be formed in the course of an amplification reaction such as a PCR reaction, but this is not necessarily the case.

Hydrolysis of RNA as carried out in the method of the invention produces adenosine monophosphate (AMP). This may be phosphorylated to adenosine triphosphate (ATP) enzymatically either directly or by way of the production of adenosine diphosphate.

ATP may be readily detected using bioluminescent systems, a particular example of which is the luciferase/luciferin detection system. Examples of the application of such detection systems are described for example in WO 96/02665.

Bioluminescent systems such as the luciferase/luciferin system do not react with the deoxyATP (dATP) which is usually added to PCR reactions in order to obtain the polymerase activity required. Thus, they will be able to distinguish between ATP produced as a result of hydrolysis of the RNA probe and any DATP which may be required to be added to the reaction mixture for other purposes.

Bioluminescent detection systems are more sensitive than fluorescent systems. Consequently, the use of RNA hydrolysis probes in analytical methods facilitates the detection of interactions at the nucleic acid level within a sample and so gives rise to enhanced methods of analysis.

In a particular embodiment, the invention provides a method for detecting the presence or amount of a target nucleic acid within a sample, said method comprising denaturing nucleic acids within a sample, contacting these with an RNA hydrolysis probe which is specific for at least a portion of said target nucleic acid so that the probe forms duplexes with the target nucleic acid; adding an enzyme which hydrolyses RNA when in double stranded form (for example as an RNA/DNA duplex) and one or more enzymes or reagents necessary to convert adenosine monophosphate produced to adenosine triphosphate; adding to the sample bioluminescent reagents which react to the presence of ATP, detecting a signal from said bioluminescent reagents and relating that to the presence or amount of the target nucleic acid sequence.

This method will frequently be carried out in the context of an amplification reaction. Thus in a further particular embodiment, the invention provides a method for detecting the presence or amount of a target nucleic acid within a sample, said method comprising conducting an amplification reaction, such as a polymerase chain reaction, in the presence of (a) an RNA probe which is specific for at least a portion of said target nucleic acid; (b) an enzyme which hydrolyses RNA when in double stranded form (for example as an RNA/DNA duplex) and (c) one or more enzymes or reagents necessary to convert adenosine monophosphate produced to adenosine triphosphate; adding to the sample bioluminescent reagents which react to the presence of ATP, detecting a signal from said bioluminescent reagents and relating that to the presence or amount of the target nucleic acid sequence.

Suitably the enzyme which hydrolyses RNA when in double stranded form ((b) above), is the polymerase used in the amplification reaction. Examples of suitable DNA polymerases which may be used in the context of the invention are thermostable polymerases such as Thermus aquaticus polymerase (Tag), Thermus thermophilus polymerase (Tth), Thermus species NH polymerase (TspNH), Thermus brockianus polymerase (Tbr) (all obtainable for example from GeneSys Limited, Farnborough, U.K.), Pyrococcus furiosus polymerase (Pfu) (obtainable from Stratagene), 9°N7 exo-DNA polymerase, and Thermococcus litoralis DNA polymerase (obtainable from New England Biolabs as VENT™ DNA polymerase).

If however, this does not hydrolyse the RNA quickly enough, for example if rapid PCR is being employed, then a suitable RNAse and in particular a DNA dependent RNAse, as would be known in the art, might also be added.

The one or more enzymes necessary to convert adenosine monophosphate produced to adenosine triphosphate ((c) above), may for example be selected from phosphoenolpyruvate synthase which produces ATP directly from AMP in the presence of phosphate and phosphoenolpyruvate, which are also added as a reagent to the reaction mixture. Alternatively, a combination of a nucleoside triphosphate-adenylate kinase and NTP will yield adenosine diphosphate (ADP), which may then be converted to ATP by inclusion or addition of an enzyme such as adenylate kinase.

Yet further examples of suitable enzymes include pyruvate phosphate dikinase such as that described by Eisaki et al, Biochim. et Biophys Acta 1431 (1999) 363-373.

Particularly suitable bioluminescent reagents, which react to the presence of ATP, include luciferin and luciferase, accompanied if necessary by a source of magnesium ions such as magnesium acetate. In the presence of ATP, these reagents produce a luminescent signal, which can be readily monitored for example using conventional luminometer devices.

In generating a signal, these reagents regenerate an AMP molecule, which in the presence of the enzymes and/or reagents of (c), will be reconverted back to ATP. Thus the signal builds up exponentially and so will be readily and rapidly detected. An example of such a system is described by Sakakibara et al., Analytical Biochemistry, 268, 94-101 (1999). This exponential rise in signal may mean that detection can be carried out directly, in circumstances where amplification reactions may previously have been required.

Suitably the bioluminescent reagents are present or added throughout the amplification reaction so that the progress of the reaction can be monitored. Generally speaking, the thermostability of reagents such as luciferase is not sufficient to allow it to be present throughout an amplification reaction and thus, it is suitably added at the end of each cycle. Such information may be used then in the quantification of the target nucleic acid sequence in the sample, using algorithms etc. which are known in the art.

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