# Patent

## Title
Polynucleotide primers

## Abstract

A polynucleotide primer comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77.

## Background
This application is a 371 filing of PCT/GB2006/001227, filed Apr. 4, 2006, which claims priority from GB 0506807.7, filed Apr. 4, 2005. These prior applications are incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to a polynucleotide, a kit comprising a polynucleotide and a method for detecting the presence or absence of mutations in a gene.
BACKGROUND ART
Epidermal Growth Factor (EGF) and its corresponding receptor (EGFR) are responsible for modulating growth of many classes of cells. Mutations of EGFR have been implicated in uncontrolled cell proliferation and tumour growth.
Although there are many examples of nucleic acid changes having potential as tumour markers, their value as clinical tools in cancer diagnosis, staging or even screening, needs to be demonstrated and two important criteria must be met. Firstly, nucleic acids of adequate yield and quality must be extracted from the clinical material; secondly, robust and accurate methods of analysis are required. For reliable tumour genotyping to be useful in disease staging any test has to be adequately validated and there should be demonstrable benefits over current methods.
A number of studies have examined the association of lung cancer and its response to gefinitib (AstraZeneca's IRESSA) with mutations in the oncogene, EGFR. However, there have been differences in the spectrum and reported frequencies of EGFR mutations. One major issue is that in order to identify a wide range of possible mutations, nucleotide sequencing of the kinase domain of the EGFR gene has been undertaken in excised tumours. The utility of this approach is limited by a number of factors, mainly:

    
    
        1. Not all of the biopsy sample is tumour
        2. Not all tumour carries the mutation
    
    


For example, as little as 10% of a specimen may be tumour, the remainder being marginal non-tumour cells. Tumours are notoriously heterogeneous in their genetic makeup and as little as 10% of the tumour cells may contain any particular genetic change. In total therefore as little as 1% of a tumour sample for genetic analysis may contain the specific variation of interest.
The detection limits for sequencing are of the order of 15-25%; that is sequencing can detect the rarer allele at levels no worse than 1 in 4 to 1 in 6. As discussed above, the prevalence of the mutation in a given tumour sample may be significantly below such levels. Current approaches require that a degree of sample enrichment be performed, namely an attempt to excise selectively the tumour material from a paraffin-embedded tissue section. This is expensive, time consuming and lacks sensitivity.
In the present invention we have now devised novel diagnostic methods for the detection of EGFR mutations based on the amplification refractory mutation system (ARMS) as disclosed in, for example, EP-A-0332435. Validated tests for four EGFR point mutations and four deletions have been developed and the tests have been applied in an investigation of the incidence of the mutations in tumours from patients. The ARMS technology is capable of selectively amplifying specific sequence variants in a background of alternative sequences.
ARMS is a simple and accurate method and has several benefits over other PCR-based mutation detection systems. Specifically, the technique does not require the use of radioisotopes nor the multiple probing of immobilised PCR amplicons nor the cloning of PCR amplicons.
ARMS avoids the need for DNA sequencing of single-strand conformation polymorphism products, a procedure that could be expected to be constrained by sequence under-representation as discussed above. Similarly, under-represented mutant sequences could go undetected using PCR in conjunction with restriction fragment length polymorphism which is limited to low cycle numbers for the PCR to avoid false positive results. Previously generated amplicons therefore have the potential to cause carry-over contamination when PCR is resumed. ARMS can be performed under conditions in which carry-over contamination is avoided, as in the present invention, allowing the use of high PCR cycle numbers and resulting in exceptionally high detection sensitivity.
DISCLOSURE OF THE INVENTION
Thus, in general terms the present invention relates to a diagnostic method for the detection of EGFR mutations in cancer, particularly lung cancer, using the amplification refractory mutation system (ARMS). The invention also relates to mutation specific primers suitable for use in the method and to diagnostic kits containing these primers.
According to one aspect of the present invention, there is provided a polynucleotide comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77. The term “the final six nucleotides” means the six nucleotides at the 3′ end of the polynucleotide.
Conveniently, the polynucleotide is less than 100 nucleotides long, preferably less than 80 nucleotides long, more preferably less than 60 nucleotides long, more preferably less than 40 nucleotides, more preferably less than 30 nucleotides long.
Preferably, the polynucleotide comprises at least 75% and more preferably 100% of the final 8, 10, 12, 14, 16, 17, 18 or 20 nucleotides, or the entirety of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77.
Advantageously, the polynucleotide further comprises a quencher group and a fluorophore group. “Fluorophore groups” are those groups which are capable of absorbing light at a first wavelength and in response emitting light at a second wavelength. A “quencher group” is a group which, when in sufficiently close proximity to a fluorophore group, is capable of preventing, or “quenching”, the emission of light from the fluorophore group. Typically, a particular type of quencher group will only work with respect to certain types of fluorophore group.
Conveniently, the quencher group and the fluorophore group are separated by a nucleotide tail sequence comprising first and second regions, the nucleotides of the first region being complementary to but in reverse order from the nucleotides of the second region, such that hybridisation of the first region to the second group results in the quencher group to be sufficiently close to the fluorophore group to quench the fluorophore group.
Preferably, the tail sequence further comprises a third region having a sequence complementary to a region of the EGFR gene.
Advantageously, the polynucleotide comprises at least the final six nucleotides of SEQ. ID NO. 3 or 10 and the tail sequence comprise SEQ. ID NO. 19.
Alternatively, the polynucleotide comprises at least the final six nucleotides of SEQ. ID NOS. 6 or 12 and the tail sequence comprises SEQ. ID NO. 20.
Alternatively, the polynucleotide comprises at least the final six nucleotides of SEQ. ID NO: 76 and the tail sequence comprises SEQ. ID NO: 77.
Conveniently, the quencher group comprises black hole quencher 1 (BHQ1) and the fluorophore group comprises FAM.
Alternatively, the quencher group comprises black hole quencher 2 (BHQ2) and the fluorophore comprises Cal Red.
Preferably, a blocking moiety is provided between the primer sequence and the tail sequence to prevent polymerase mediated chain extension of the tail sequence. A preferred blocking moiety is a hexethylene glycol (HEG) monomer.
According to another aspect of the present invention, there is provided a kit comprising at least a pair of polynucleotides, wherein the pair of polynucleotides comprises at least four or five of the final six nucleotides of one of the following pairs of primer sequences, respectively, or sequences complementary thereto: SEQ. ID NO. 1 and SEQ. ID NO. 15, or SEQ. ID NO. 2 and SEQ. ID NO 15, or SEQ. ID NO. 3 and SEQ. ID NO. 15, or SEQ. ID NO. 4 and SEQ. ID NO. 15, or SEQ. ID NO. 5 and SEQ. ID NO. 15, or SEQ. ID NO. 6 and SEQ. ID NO. 16, or SEQ. ID NO. 7 and SEQ. ID NO. 17, or SEQ. ID NO. 8 and SEQ. ID NO. 18, or SEQ. ID NO. 9 and SEQ. ID NO. 18, or SEQ. ID NO. 10 and SEQ. ID NO. 15, or SEQ. ID NO. 11 and SEQ. ID NO. 15 or SEQ. ID NO. 12 and SEQ. ID NO. 16, or SEQ. ID NO. 13 and SEQ. ID NO. 17, or SEQ. ID NO. 14 and SEQ. ID NO. 18 or SEQ. ID NO. 21 and SEQ. ID NO. 15, or SEQ. ID NO. 22 and SEQ. ID NO. 24, or SEQ. ID NO. 23 and SEQ. ID NO. 24, or SEQ. ID NO. 25 and SEQ. ID NO. 41, or SEQ. ID NO. 26 and SEQ. ID NO. 41, or SEQ. ID NO. 27 and SEQ. ID NO. 41, or SEQ. ID NO. 28 and SEQ. ID NO. 41, or SEQ. ID NO. 29 and SEQ. ID NO. 42, or SEQ. ID NO. 30 and SEQ. ID NO. 43, or SEQ. ID NO. 31 and SEQ. ID NO. 44, or SEQ. ID NO. 32 and SEQ. ID NO. 44, or SEQ. ID NO. 33 and SEQ. ID NO. 41, or SEQ. ID NO. 34 and SEQ. ID NO. 45, or SEQ. ID NO. 35 and SEQ. ID NO. 41, or SEQ. ID NO. 36 and SEQ. ID NO. 41, or SEQ. ID NO. 37 and SEQ. ID NO. 42, or SEQ. ID NO. 38 and SEQ. ID NO. 43, or SEQ. ID NO. 39 and SEQ. ID NO. 44, or SEQ. ID NO. 40 and SEQ. ID NO. 45, or SEQ. ID NO. 74 and SEQ. ID NO. 76 or SEQ. ID NO. 75 and SEQ. ID NO. 76. For example, the pair of polynucleotides comprises a first polynucleotide consisting of the final six nucleotides of SEQ ID NO. 1 and a second polynucleotide consisting of the final six nucleotides of SEQ ID NO. 15.
According to a further aspect of the present invention, there is provided a kit comprising at least a set of three polynucleotides wherein the set of three polynucleotides comprises at least four or five of the final six nucleotides of one of the following sets of three primer sequences, respectively, or sequences complementary thereto: SEQ. ID NOS. 1, 10 and 15, or SEQ. ID NOS. 2, 10 and 15, or SEQ. ID NOS. 3, 10 and 15, or SEQ. ID NOS. 4, 11 and 15, or SEQ. ID NOS. 5, 11 and 15, or SEQ. ID NOS. 6, 12 and 16, or SEQ. ID NOS. 7, 13 and 17, or SEQ. ID NOS. 8, 14 and 18, or SEQ. ID NOS. 9, 14 and 18, or SEQ. ID NOS. 21, 10 and 15, or SEQ. ID NOS. 22, 23 and 24, or SEQ. ID NOS. 25, 35 and 41, or SEQ. ID NOS. 26, 35 and 41, or SEQ. ID NOS. 27, 36 and 41, or SEQ. ID NOS. 28, 36 and 41, or SEQ. ID NOS. 29, 32 and 42, or SEQ. ID NOS. 30, 38 and 43, or SEQ. ID NOS. 31, 39 and 44, or SEQ. ID NOS. 32, 39 and 44, or SEQ. ID NOS. 33, 35 and 41, or SEQ. ID NOS. 34, 40 and 45 or SEQ. ID NOS. 74, 75, 76. For example, the set of three polynucleotides comprises a first polynucleotide consisting of the final six nucleotides of SEQ ID NO. 1, a second polynucleotide consisting of the final six nucleotides of SEQ IS NO. 10 and a third polynucleotide consisting of the final six nucleotides of SEQ ID NO. 15.
Conveniently the polynucleotides in the kit are as described above.
Preferably, the kit further comprises nucleotide triphosphates, a polymerisation enzyme and/or a buffer solution.
According to another aspect of the present invention, there is provided the use of a polynucleotide or a kit as described above or a polynucleotide comprising four or five of the final six nucleotides of SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 or sequences complementary thereto for detecting a mutation in a nucleic acid sample containing at least a fragment of the EGFR gene.
Advantageously, the fragment of the EGFR gene in the nucleic acid sample is at least 10 nucleotides long, preferably 20 nucleotides long, more preferably 30 nucleotides long and more preferably 40 nucleotides long.
According to a further aspect of the present invention, there is provided a method of detecting the presence or absence of a mutation in the EGFR gene comprising the steps of:

    
    
        a) mixing a nucleic acid sample comprising at least a fragment of the EGFR gene with a polynucleotide complementary to a region of the fragment of the EGFR gene; and
        b) detecting hybridisation of the polynucleotide to the nucleic acid sample wherein hybridisation indicates the presence or absence of a mutation.
    
    


Advantageously, the polynucleotide is a polynucleotide as described above and comprises at least for or five of the final six nucleotides of SEQ. ID NOS. 1 to 9, 21, 22, 25 to 34 or 75 or sequences complementary thereto and step b) indicates the presence of a mutation.
Preferably, the polynucleotide is a polynucleotide as described above and comprises at least for or five of the final six nucleotides of SEQ. ID NOS. 10 to 14, 23, 35 to 40 or 74 or sequences complementary thereto; and step b) indicates the absence of a mutation.
Conveniently, the method further comprises the step of prior to step a), amplifying the number of copies of the fragment of the EGFR gene using thermal cycling nucleic acid amplification, preferably PCR.
Preferably step b) comprises carrying out DNA polymerisation using the polynucleotide as a first primer and detecting the extension product of polymerisation.
Advantageously, the method step b) comprises the step of mixing the nucleic acid sample and the polynucleotide with a second primer which corresponds to a region of the fragment of the EGFR sequence downstream of the region to which the polynucleotide is complementary and carrying out PCR on the mixture.
Conveniently, the second primer comprises: SEQ. ID NO. 15 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 1 to 5, 10, 11 or 21; SEQ. ID NO. 16 and the polynucleotide comprises at least the four or five final six nucleotides of SEQ. ID NOS. 6 or 12; SEQ. ID NO. 17 and the polynucleotide comprises at least the four or five final six nucleotides of SEQ. ID NOS. 7 or 13; or SEQ. ID NO. 18 and the polynucleotide comprises at least the four or five final six nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO. 24 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 31, 32 or 39; or SEQ. ID NO. 45 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 34 or 40; SEQ. ID NO. 76 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NO. 74 or 75.
Preferably step a) comprises the step of mixing the nucleic acid sample with a pair of a mutation specific polynucleotide and a wild-type specific polynucleotide, the pair being selected from at least four or five of the final six sequences of: SEQ. ID NO: 1 and SEQ. ID NO: 10; SEQ. ID NO: 2 and SEQ. ID NO: 10; SEQ. ID NO: 3 and SEQ. ID NO: 10; SEQ. ID NO: 4 and SEQ. ID NO: 11; SEQ. ID NO: 5 and SEQ. ID NO: 11; SEQ. ID NO: 6 and SEQ. ID NO: 12; SEQ. ID NO: 7 and SEQ. ID NO: 13; SEQ. ID NO: 8 and SEQ. ID NO: 14; SEQ. ID NO: 9 and SEQ. ID NO: 14; SEQ. ID NO: 21 and SEQ. ID NO: 10; SEQ. ID NO: 22 and SEQ. ID NO: 23; SEQ. ID NO: 25 and SEQ. ID NO: 35; SEQ. ID NO: 26 and SEQ. ID NO: 35; SEQ. ID NO: 27 and SEQ. ID NO: 36; SEQ. ID NO: 28 and SEQ. ID NO: 36; SEQ. ID NO: 29 and SEQ. ID NO: 37; SEQ. ID NO: 30 and SEQ. ID NO: 38; SEQ. ID NO: 31 and SEQ. ID NO: 39; SEQ. ID NO: 32 and SEQ. ID NO: 39; SEQ. ID NO: 33 and SEQ. ID NO: 35; SEQ. ID NO: 34 and SEQ. ID NO: 40; or SEQ. ID NO. 74 and SEQ. ID NO. 75.
Advantageously the nucleic acid sample comprises wild-type sequences and mutated sequences and further comprising step c) wherein the number of amplification cycles required to amplify the wild-type sequences to a predetermined quantity is compared with the number of amplification cycles required to amplify the mutated sequences to the predetermined quantity thereby providing an indication of the ratio of the wild type sequences to mutated sequences in the sample.
Conveniently the nucleic acid sample comprises a portion of tumourous tissue and a portion of non-tumourous tissue and wherein step c) further comprises the step determining the ratio of tumourous tissue to non-tumourous tissue in the sample.
Preferably the method further comprises the step of, prior to step a), enriching the nucleic acid sample to increase the ratio of tumourous tissue to non-tumourous tissue in the sample.
Preferably step b) comprises detecting if amplification of at least a portion of the EGFR gene occurs.
Advantageously the polynucleotide comprises a quencher group and a fluorophore group and step b) comprises exposing the mixture to light of a wavelength to which the fluorophore is responsive in the absence of the quencher group and detecting light at the wavelength emitted by the fluorophore group in the absence of the quencher group.
Where reference is made in the specification to “at least four or five of the final six sequences” of a reference sequence, this means that, of the six nucleotides in the reference sequence, either one or two of the nucleotides may be missing or replaced with a different nucleotide. Of course, in some embodiments, the sequence comprises all six of the nucleotides of the reference sequence.
In preferred embodiments of the present invention, reference to “EGFR” is to the human EGFR gene available as accession number NC—000007 in Homo sapiens chromosome 7, region 55054219 . . . 55242525 as on 30 Mar. 2006. In some other embodiments, reference is to the sequence as on 4 Mar. 2005. Both sequences are incorporated herein by reference. In some embodiments, the EGFR gene is not identical to these sequences but is at least 90% identical to either or both of the sequences.
A reference in this specification is made to a percentage of a polynucleotide compared with a reference polynucleotide, this can be determined by algorithms known in the art.
For example the percentage identity between two sequences can be determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402) using default parameters.



BRIEF DESCRIPTION OF DRAWINGS
In order that the present invention may be more readily understood and so that further features thereof may be appreciated, embodiments of the invention will now be described, by way of example, with reference to the accompanying figures in which:
FIG. 1 is a graph showing the results of PCR amplification carried out on a sample in the presence of a primer of the present invention and control primers;
FIG. 2 is a graph showing the results of PCR amplification carried out on a sample in the presence of a primer specific for del S752-I759 (SEQ. ID NO. 5) and a corresponding wild-type specific primer;
FIG. 3 is a graph showing the results of PCR amplification carried out on a sample in the presence of a primer specific for the L858R mutation (SEQ. ID NO. 6) and a corresponding wild-type specific primer;
FIG. 4 is a graph (lower) showing the results of PCR amplification carried out on a sample in the presence of a primer specific to a mutation and a corresponding wild-type specific primer, and a graph (upper) showing the results of sequencing of the sample;
FIG. 5 is a graph (lower) showing the results of PCR amplification carried out on a sample in the presence of a primer specific to a mutation and a corresponding wild-type specific primer, and a graph (upper) showing the results of sequencing of the sample; and
FIG. 6 is a graph showing the results of PCR amplification carried out on a set of samples, in the presence of a primer specific to a mutation; and
FIG. 7 is a graph showing the results of PCR amplification, using control primers, of the samples analysed in the results shown in FIG. 6.



DETAILED DESCRIPTION
In general terms, the present invention provides a diagnostic method of the detection of EGFR mutations in cancer, which method comprises contacting a test sample of nucleic acid with a diagnostic primer for a EGFR mutation in the presence of appropriate nucleotide triphosphates and an agent for polymerisation, such that the diagnostic primer is efficiently extended only when a EGFR mutation is present in the sample; and detecting the presence or absence of a EGFR mutation by reference to the presence or absence of a diagnostic primer extension product.
There are disclosed herein, primers (SEQ. ID NOS: 1 to 9, 21, 22 and 75) which can be used in the method of the invention.
Each of the diagnostic primers detects the presence or absence of one of the following EGFR mutations:

    
    
        1) del E746-A750 (two different mutations exist)
        2) del L747-T751 insS
        3) del L747-P753 insS
        4) del S752-I759
        5) Exon 21 L858R,
        6) Exon 21 L861Q,
        7) Exon 18 G719C
        8) Exon 18 G719S
        9) Exon 20 T790M
    
    


Mutations 1) to 8) confer sensitivity of an individual to gefinitib whereas mutation 9) confers resistance to gefinitib.
In some embodiments, diagnostic primers specific for the corresponding mutations on the opposite DNA strand are used (SEQ. ID NOS: 25 to 34).
The test sample of nucleic acid is conveniently a sample of blood, faeces, sputum, colonic lavage, bronchial lavage or other body fluid, or tissue obtained from an individual. The individual is conveniently human, preferably Homo sapiens. It will be appreciated that the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample. That is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique such as PCR or whole genome amplification (WGA) before use in the method of the invention.
Any convenient enzyme for polymerisation may be used provided that it does not affect the ability of the DNA polymerase to discriminate between normal and mutant template sequences to any significant extent. Examples of convenient enzymes include thermostable enzymes which have no significant 3′-5′ exonuclease activity, for example Taq DNA polymerase, particularly “Ampli Taq Gold”TM DNA polymerase (PE Applied Biosystems), Stoffel fragment, or other appropriately N-terminal deleted modifications of Taq or Tth (Thermus thermophilus) DNA polymerases.
There are disclosed herein primers for the above EGFR point mutations which have been shown to detect the specific mutations reliably and robustly. Therefore in a further aspect of the invention we provide diagnostic primers comprising SEQ. ID NOS: 1 to 9, 21, 22, 25 to 34 or 75 and derivatives thereof wherein 6-8 of the nucleotides at the 3′ end are identical to the sequences and wherein up to 10, such as up to 8, 6, 4, 2, 1, of the remaining nucleotides are optionally varied without significantly affecting the properties of the diagnostic primer. Conveniently, the sequence of the diagnostic primer is exactly as shown in any one of SEQ. ID NOS: 1 to 9, 21, 22, 25 to 34 or 75.
It is to be appreciated that alternative versions of the above described diagnostic methods are configured so that extension of the diagnostic primer indicates the absence of the EGFR mutation. For example, in the embodiments the primers comprise 6-8 nucleotides from the 3′ end of any one of SEQ. ID NOS. 10 to 14, 23 or 74. In some embodiments, primers specific for the wild type sequences present the corresponding section of the complementary DNA strand are used (SEQ. ID NOS. 35 to 40).
In many embodiments, it is convenient to use a diagnostic primer of the invention with a further amplification primer in one or more cycles of PCR amplification. A convenient example of this aspect is set out European patent number EP-A-1-0332435. The further amplification primer is either a forward or a reverse common primer. Examples of such common primers are SEQ. ID NOS: 15 to 18, 24 and 76 and, to be used in conjunction with the primers for the complementary strand, SEQ. ID NOS: 41 to 45.
Any convenient control primer pair may be used. Control primers from an unrelated region of the genome, namely part of the human albumen gene, are used herein.
The diagnostic methods of the invention as outlined above are conveniently effected in one or more reaction vessels in some embodiments. Where more than one diagnostic mutation is to be assayed, the diagnostic primer (and corresponding amplification primer) are provided in individual tubes i.e. one tube per mutation in certain embodiments. Alternatively, the reactions are multiplexed, that is to say that all the diagnostic primers and amplification primers are in one tube (see EP-A-1-0332435) in other embodiments.
A variety of methods may be used to detect the presence or absence of diagnostic primer extension products and/or amplification products. These will be apparent to the person skilled in the art of nucleic aid detection procedures. Preferred methods avoid the need for radio-labelled reagents. Particular detection methods include “TaqMan”TM product detection, for example as described in U.S. Pat. No. 5,487,972 & U.S. Pat. No. 5,210,015; Scorpions (WO-A-99/066071), which has particular benefits because specific diagnostic primers may be linked to a specific detector fluorophore, allowing the detection of multiple mutation targets from the same region of the genome.
Conveniently, real-time detection is employed which allows the quantitation of the mutation(s) within the sample. More specifically, the number of cycles required to amplify a DNA sample to a predetermined level using primers specific for the wild-type sequences is compared with the number of cycles required using primers specific for a mutant sequence. By the use of this comparison and by reference to control amplifications containing standardised proportions of mutant target, the ratio of the amount of DNA in the sample containing the mutation relative to the amount of wild type DNA is quantified. Of course this only relates to the sample as presented and does not compensate for low tumour representation within the sample. For this reason, it is preferred to combine this quantitative allele specific approach with a method to estimate the level of tumour within the sample. Alternatively, the quantitative allele specific approach is combined with a method to enrich the sample by specifically excising the tumour material from the overall sample.
In some embodiments, one or more of the diagnostic primers of the invention is conveniently packaged with instructions for use in the method of the invention and appropriate packaging and sold as a kit. The kits conveniently include one or more of the following: appropriate nucleotide triphosphates, for example dATP, dCTP, dGTP, dTTP, a suitable polymerase as previously described, and a buffer solution.
The invention will now be illustrated but not limited by reference to the following Examples.
Materials And Methods
Construction of Synthetic Templates
In the absence of validated and uniform genomic material that carries each of the mutations, it was necessary to construct synthetic templates. For EGFR deletion tests this was done using long synthetic overlapping oligonucleotides, each comprising approximately half the desired amplicon. The sequences of these long oligonucleotides are shown in Table 1.









TABLE 1







SEQ.


Name
Sequence
ID NO.







EGFRDEL 15
AAAATTCCCGTCGCTATCAAAACATCTCCGAA
47


CONFOR
AGCCAACAAGGAAATCCTCGATGTGAGTTTCT




GCTTTGCTGTGTGGGGGTCCATGGCTCTGAAC




CTCA



 


EGFRDEL 15
AACATTTAGGATGTGGAGATGAGCAGGGTCTA
48


CONREV
GAGCAGAGCAGCTGCCAGACATGAGAAAAGGT




GGGCCTGAGGTTCAGAGCCATGGACC



 


EGFRDEL 18
CGCTATCAAGGAATCGAAAGCCAACAAGGAAA
49


CONFOR
TCCTCGATGTGAGTTTCTGCTTTGCTGTGTGG




GGGTCCATGGCTCTGAACCTCAGGCCCACCTT




TTCT



 


EGFRDEL 18
AGAAAGACATAGAAAGTGAACATTTAGGATGT
50


CONREV
GGAGATGAGCAGGGTCTAGAGCAGAGCAGCTG




CCAGACATGAGAAAAGGTGGGCCTGAGGT



 


EGFRDEL 24
GCTATCAAGGAATTAAGAGAAGCAACACTCGA
51


CONFOR
TGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCA




TGGCTCTGAACCTCAGGCCCACCTTTTCTCAT




GTCT



 


EGFRDEL 24
AGAAAGACATAGAAAGTGAACATTTAGGATGT
52


CONREV
GGAGATGAGCAGGGTCTAGAGCAGAGCAGCTG




CCAGACATGAGAAAAGGTGGGC



 


EGFRDEL 12
AATTCCCGTCGCTATCAAGGAACCATCTCCGA
53


CONFOR
AAGCCAACAAGGAAATCCTCGATGTGAGTTTC




TGCTTTGCTGTGTGGGGGTCCATGGCTCTGAA




CCTC



 


EGFRDEL 12
GTGAACATTTAGGATGTGGAGATGAGCAGGGT
54


CONREV
CTAGAGCAGAGCAGCTGCCAGACATGAGAAAA




GGTGGGCCTGAGGTTCAGAGCCATGGACC









In order to construct the targets, the two overlapping 1⁄2-amplicons were mixed in equimolar concentrations in the presence of polymerase, buffer and dNTPs. The primer mix was then incubated for 25 cycles of 1 minute each with an increment of 1° C. per cycle from 50° C. to 75° C. Synthetic templates were subsequently diluted 1 in 1 million into genomic DNA for use as controls in PCR reactions.
For SNP tests synthetic cassettes were produced using PCR to incorporate required mutations into two half cassettes. Half cassettes were then mixed in equimolar concentrations and used as a template for the construction of full length cassettes containing the required SNP target mutation. Specific primer sequences for the construction of cassettes are shown in Table 2.









TABLE 2







SEQ.


Name
Sequence
ID NO.







EGFR Ex18 AU
CGCCATGCACAACTTCCCTAC
55


 


EGFR Ex18 AL
TCCAGAATTTAATGATGCTGCGTCT
56


 


EGER Ex18 ML
GAACGCACCGGAGCACA
57


 


EGFR Ex18 BU
TTGGTGACATGTTGGTACATCCATC
58


 


EGFR Ex18 MU
TTCAAAAAGATCAAAGTGCTGTGC
59


 


EGER Ex18 BL
CGTTAACTGGCAATTGTGAGATGGT
60


 


EGFR Ex21 AU
AGTCCAGTAAGTTCAAGCCCAGGTC
61


 


EGFR Ex21 AL
GTTCCTTAGGTGTCCTTGACAGCAG
62


 


EGFR Ex21
CCAGCAGTTTGGCCCGC
63


L858R ML




 


EGFR Ex21 BU
CAGAGATTTCAATTGCAGCGAGATT
64


 


EGFR Ex21
AGATCACAGATTTTGGGCGGG
65


L858R MU




 


EGFR Ex21 BL
TAGGTTTCTGAGCACCCTCTGTGTC
66


 


EGFR Ex21
TCTCTTCCGCACCCAGCTGT
67


L861Q ML




 


EGFR Ex21
TTGGGCTGGCCAAACAGC
68


L861Q MU




 


EGFR Ex20
ACCGTGCAGCTCATCATGC
46


T790M MU




 


EGFR Ex20
GCTGTGAAATACCTGGCTTGTTGTT
69


T790M BL




 


EGFR Ex20
GAAGGGCATGAGCTGCATG
70


T790M BU




 


EGFR Ex20
CCAGGCAGCCTTTAGTCACTGTAGA
71


T790M ML




 


EGFR Ex20
GGCCTCTCTGTCATGGGGAAT
72


T790M AU




 


EGFR Ex20
ACCTGCTCCACTCCACCACTATCAC
73


T790M AL









Variant Specific PCR

Specific primer sequences for each ARMS reaction and control are shown in Tables 3 and 4.
Reactions were performed in 0.2 ml vessels (single tubes, strips or plates). Reactions (25 μl) typically contained:

    
    
        250 nM diagnostic primer
        250 nM reverse primer
        250 nM each of control primers (where used)
        200 μM each dNTP
        10 mM Tris-HCl (pH 8.3)
        50 mM KCl
        2.5-4 mM MgCl2
    
    


Some reactions performed better in the presence of Qiagen Multiplex Reaction buffer that contains a proprietary mixture of Ammonium Sulphate, MgCl2, and volume excluders such as Poly (ethylene glycol) MW 8000, and/or Dextran (MW 50,000); see US-A-2004-0115712.
For Real Time Quantitative PCR, YO-PRO-1 (Molecular Probes) was included at a final concentration of 1 μM. This dye binds double-stranded DNA and fluoresces with high efficiency, enabling a general detection of PCR product in a reaction. Alternatively, and more preferred, we used Scorpions (a molecule that combines a primer and fluorogenic probe), which offers specific detection of target amplicons.







TABLE 3







Allele Specific primers (1)











WILD TYPE PRIMERS
MUTANT PRIMERS
COMMON PRIMERS


















SEQ


SEQ


SEQ





ID


ID


ID



Test
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE





del L747-
EGFR
10
CGT CGC TAT
EGFR DEL
 1
CCC GTC GCT
EGFR
15
GAG ATG AGC


T751 insS
WTA

CAA GGA ATT
12

ATC AAG GA
EX19 REV

AGG GTC TAG





AAG AGA AGC


CCA


AGC AGA G


 


del E746-
EGFR
10
CGT CGC TAT
EGFR DEL
 2
TTA AAA TTC
EGFR
15
GAG ATG AGC


A750
WTA

CCA GGA ATT
15

CCG TCG CTA
EX19 REV

AGG GTC TAG





AAG AGA AGC


TCA AAA C


AGC AGA G


 


del E746-
EGFR
19
FAM CCGCGG
EGFR DEL
19
CAL RED CCG
EGFR
15
GAG ATG AGC


A750
WTA
and
GATTTCCTTG
15
and
CGG GATTTCC
EX19 REV

AGG GTC TAG


Scorpions
Scorpion
10
TTGGCTTTCG
Scorpions
 3
TTGTTGGCTT


AGC AGA G





CCGCGG


TCG CCGCGG








BHQ1 HEG


BHQ2 HEG








CGTCGCTATCA


TTAAAATTCCC








AGGAATTAAGA


GTCGCTATCAA








GAAGC


AAC





 


del L747-
EGFR
11
GAG AG CA
EGFR DEL
 4
CCG TCG CTA
EGFR
15
GAG ATG AGC


P753 insS
WT

CAT CTC CGA
18

TCA AGG AAT
EX19 REV

AGG GTC TAG





AAG CC


CGA


AGC AGA G


 


del S752-
EGFR
11
GAG AG CA
EGFR DEL
 5
CAA GGA ATT
EGFR
15
GAG ATG AGC


I759
WT

CAT CTC CGA
24

AAG AGA AGC
EX19 REV

AGG GTC TAG





AAG CC


AAC ACT CGA


AGC AGA G


 


Exon 21
EGFR
12
CAT GTC AAG
EGFR
 6
CAT GTC AAG
EGFR
16
GCT GAC CTA


L858R
EX21

ATC ACA GAT
EX21

ATC ACA GAT
EX21

AAG CCA CCT



L858R

TTT GGC CT
L858R

TTT GGG AG
L858R

CCT TAC T



WT


MUT


COM




 


Exon 21
EGFR
20
FAM CCGCGG
EGFR
20
CAL RED CCG
EGFR
16
GCT GAC CTA


L858R
EX21
and
ATTCTTTCTCT
EX21
and
CGG ATTCTTT
EX21

AAG CCA CCT


Scorpions
L858R
12
TCCGCACCCCC
L858R
 6
CTCTTCCGCA
L858R

CCT TAC T



WT

GCGG BHQ1
MUT

CCC CCGCGG
COM





Scorpion

HEG CATGTCA
Scorpions

BHQ2 HEG CA








AGATCACAGAT


TGTCAAGATCA








TTTGGCCT


CAGATTTTGGG











AG





 


Exon 21
EGFR
13
TTT CTC TTC
EGFR
 7
TTT CTC TTC
EGFR
17
CTG TTT CAG


L861Q
EX21

CGC ACC CAC
EX21

CGC ACC CAG
EX21

GGC ATG AAC



L861Q

CA
L861Q

AT
L861Q

TAC TTG G



WT


MUT


COM




 


Exon 18
EGFR
14
CTG AAT TCA
EGFR
 8
CTG AAT TCA
EGFR
18
CCT TTG GTC


G719C
EX18

AAA AGA TCA
EX18

AAA AGA TCA
EX18

TGT GAA TTG



G719C

AAG TGC TCG
G719C

AAG TGC CGT
COM

GTC TCA C



WT


MUT
















TABLE 4







Allele Specific primers (2)











WILD TYPE PRIMERS
MUTANT PRIMERS
COMMON PRIMERS


















SEQ


SEQ


SEQ





ID


ID


ID



Test
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE





Exon 18
EGFR
14
CTG AAT TCA
EGFR
 9
CTG AAT TCA
EGFR
18
CCT TTG GTC


G719S
EX18

AAA AGA TCA
EX18

AAA AGA TCA
EX18

TGT GAA TTG



G719S

AAG TGC TCG
G719S

AAG TGC CGA
COM

GTC TCA C



WT


MUT







 


EGFR del
EGFR
10
CGT CGC TAT
EGFR del
21
TTAAAATTCCC
EGFR
15
GAG ATG AGC


15 2
WTA

CAA GGA ATT
15 2 For

GTCGCTATCAA
EX19

AGG GTC TAG





AAG AGA AGC


GACA
REV

AGC AGA G


 


EGFR
EGFR
23
ACCTCCACCGT
EGFR
22
ACCTCCACCGT
EGFR
24
ATGGCAAACTC


T790M
T790M

GCAGCTCATA
T790M

GCAGCTCATC
T790M

TTGCTATCCCA



FC

AC
FT

CT
Reverse

GGA


 


EGFR
T790M
74
TCCACCGTGCA
T790M
75
TCCACCGTGCA
EGFR
76
TTGTCTTTGTG


T790M
A

GCTCATCTC
B

GCTCATCTT
T790M

TTCCCGGACAT









Reverse II




 


EGFR
T790M
74
TCCACCGTGCA
T790M
75
TCCACCGTGCA
EGRR
76
FAM-CCGCGGC


T790M
A

GCTCATCTC
B

GCTCATCTT
T790M
and
TCATGCCCTTC









Reverse II
77
GGCTCCGCGG-









Scorpions

DABCYL-HEG-











TTGTCTTTGTG











TTCCCGGAC











AT-3′









PCRs were performed in Real Time PCR cyclers (Mx4000 and Mx3000P from Stratagene), using standard conditions:

    
    
        95° C. for 10-15 minutes to activate the hot-start enzyme, followed by up to 50 cycles of:
        95° C., 60 s
        65° C., 60 s—Deletion tests
    
    


For SNP tests, the anneal/extend step was modified to 60° C., for 60 s.
Measurement of Extracted DNA
The amount of DNA extracted from each tumour sample was measured by real time amplification of a control locus. Standard curves for this reaction were generated using known concentrations of high quality DNA extracted from cell lines (ECACC, Wiltshire, UK).
Sequencing of Controls and Positive Samples
Sequencing was performed on amplicons generated from exon specific primers, by standard cycle sequencing, using big dye terminators. Reactions were separated using an ABI 3100 capillary sequencing instrument, according to the manufacturer's instructions.
Results and Discussion
ARMS Tests Development and Validation
ARMS tests specific for each of the deletions and SNPs were tested against:

    
    
        1. “normal” genomic DNA (gDNA)
        2. mismatched synthetic targets
        3. synthetic target diluted in buffer
        4. synthetic target diluted in gDNA (to mimic the tumour situation)
        5. No template (water instead of template)
    
    


Reactions included the fluorescent DNA binding dye YO-PRO-1 and were monitored in real time. In such reactions, the point at which amplification becomes visible above baseline (known as the Ct) is an indicator of the quantity of target.
Primers which are specific for the same mutations but on the complementary DNA strand have also been developed and these are shown in Tables 5 and 6.







TABLE 5







Allele Specific primers for Reverse Strand (I)











WILD TYPE PRIMERS
MUTANT PRIMERS
COMMON PRIMERS


















SEQ


SEQ


SEQ





ID


ID


ID



Test
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE





del L747-
EGRF WT
35
GGCTTTCGGAGA
EGFR del
25
CTTGTTGGCTTT
EGFR
41
CTGGTAACATC


T751 insS
Rev del

TGTTGCTTCTC
12 rev

CGGAGATGGT
Ex 19

CACCCAGATCA



15 + 12





For

CTG


 


del E746-
EGFR WT
35
GGCTTTCGGAGA
EGFR del
26
TTGTTGGCTTTC
EGFR
41
CTGGTAACATC


A750
Rev del

TGTTGCTTCTC
15 1 rev

GGAGATGTTTTG
Ex 19

CACCCAGATCA



15 + 12





For

CTG


 


deL L747-
EGFR WT
36
GCTTCTCTTAAT
EGFR del
27
GGATTTCCTTGT
EGFR
41
CTGGTAACATC


P753 insS
Rev

TCCTTGATAGCG
18 rev

TGGCTTTCGAT
Ex 19

CACCCAGATCA



18 + 24

ACG



For

CTG


 


deL S752-
EGFR WT
36
GCTTCTCTTAAT
EGFR del
28
CAAAGCAGAAAC
EGFR
41
CTGGTAACATC


I759
Rev

TCCTTGATAGCG
24 Rev

TCACATCGAGTG
Ex 19

CACCCAGATCA



18 + 24

ACG



For

CTG


 


Exon 21
EGFR
37
CTCTTCCGCACC
EGFR
29
CTCTTCCGCACC
EGFR
42
TTCCCATGATG


L858R
L858R

CAGCAGTTTGGT
L858R

CAGCAGTTTGGC
L858R

ATCTGTCCCTC



RT

CA
RG

AC
Forward

ACAGCA


 


Exon 21
EGFR
38
ATCACAGATTTT
EGFR
30
ATCACAGATTTT
EGFR
43
GAGCTCACCCA


L861Q
L861Q

GGGCTGGCCAAT
L861Q

GGGCTGGCCAAT
L861Q

GAATGTCTGGA



FA

CA
FT

AA
Reverse

GAGCAT


 


Exon 18
EGFR
39
TATACACCGTGC
EGFR
31
TATACACCGTGC
G719
44
GGGCTGAGGTG


G719C
G719 R

CGAACGCACCGG
G719C

CGAACGCACCGG
Forward

ACCCTTGTCTC



WT

AGAC
RT

AACA


TGTGTT


 


Exon 18
EGFR
39
TATACACCGTGC
EGFR
32
TATACACCGTGC
G719
44
GGGCTGAGGTG


G719S
G719 R

CGAACGCACCGG
G719S

CGAACGCACCGG
Forward

ACCCTTGTCTC



WT

AGAC
RA

ATCT


TGTGTT


 


EGFR del
EGFR WT
35
GGCTTTCGGAGA
EGFR del
33
TTGTTGGCTTTC
EGFR
41
CTGGTAACATC


15 2
Rev del

TGTTGCTTCTC
15 2 rev

GGAGATGTCT
Ex 19

CACCCAGATCA


Reverse
15 + 12





For

CTG
















TABLE 6







Allele Specific primers for Reverse Strand (II)











WILD TYPE PRIMERS
MUTANT PRIMERS
COMMON PRIMERS


















SEQ


SEQ


SEQ





ID


ID


ID



Test
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE
NAME
NO.
SEQUENCE





EGFR
EGFR
40
AGCCGAAGGGC
EGFR
34
AGCCGAAGGG
EGFR
45
GCACAGCTTT


T790M
T790M

ATGAGCTTCA
T790M

CATGAGCTG
T790M

TCCTCCATGA


Reverse
RT




AG
Forward

GTACG



WT









Example 1
The mutation specific primer EGFR Del 12 was tested against matched synthetic target and three mismatched mutant synthetic targets (del 15 (1), del 18 and del 24), as well as normal DNA and a water control. The results are shown in FIG. 1.
It is clear that in this reaction the primer shows absolute specificity for its own target and does not detect wild type sequences nor any of the other deletion mutations found in the same genomic region.
Example 2
Use of ARMS Tests on Tumour Samples
Forty-two DNA samples from Non-Small Cell Lung Cancers and cell lines were analysed using each of the mutation specific primers SEQ. ID NOS: 1 to 9. Tumour DNA had been extracted from paraffin embedded tissue on slides.
A number of samples were found to be positive using this method: nine for the L858R SNP and one for the 15 base pair deletion del E746-A750.
Confirmatory sequencing was performed for the region surrounding the putative mutations.
FIG. 2 shows a typical negative result for a batch of 8 samples tested with the del S752-I759 specific primers. It is clear that the wild type specific reactions perform efficiently while the mutation specific primers (SEQ. ID NO: 5) do not amplify indicating the absence of this mutation in these samples.
FIG. 3 shows the L858R analysis for 8 positive samples. In each case, the reaction using the mutation primers (SEQ. ID NO: 6) is positive as is the wild type reaction. The fact that the mutant reaction appears several cycles later than the corresponding wild type reaction indicates that the mutation is not the majority sequence in the samples.
FIG. 4 shows more detailed analysis of the allele specific PCR analysis, combined with sequencing of the same exon from this sample. In this sample, the sequencing approach would have failed to detect the mutation that was clearly identified by the ARMS approach (no peak is visible at the circled position).
FIG. 5 shows more detailed analysis of the allele specific PCR analysis, combined with sequencing of the same exon from this sample. In this sample, the sequencing approach could have detected the mutation that was clearly identified by the ARMS approach (a very weak peak is visible at the circled position). The differential (ΔCt) between the wild type and mutant specific reactions was smaller in this sample, indicating that the mutation was relatively more prevalent than in the sample whose analysis is shown in FIG. 4.
Example 3
FIG. 6 shows the results of PCR amplification of samples containing DNA with, and without the T790M mutation. Five DNA samples were provided, each comprising 0%, 1%, 10%, 50% or 100% DNA with the T790M mutation. The samples were amplified using the mutant primer of SEQ. ID NO. 75 and the reverse Scorpion primer of SEQ. ID NOS. 76 and 77. The results show that the mutant primer was specific for DNA having the T780M mutation because the sample without any DNA having the mutation showed no amplification. Furthermore, detection was shown to be quantitative in that the Ct (threshold cycle) was related directly to the relative amount of matched target, even in the presence of excess unmatched target.
FIG. 7 shows the results of a control assay carried out on the samples which were tested and reported in FIG. 6. PCR amplification was carried out using control primers. FIG. 7 confirms that each sample had the same total amount of target since the threshold cycles for each sample were the same in this control amplification.

## Claims

1. An isolated EGFR mutation diagnostic primer comprising one of the following primer sequences, or the sequence complementary to one of the following primer sequences: SEQ. ID NOS. 1, 2, 4, 5, 6, 19, 20, 21 or 75, wherein the primer further comprises a quencher group and a fluorophore group.

2. A primer according to claim 1 wherein the quencher group and the fluorophore group are separated by a nucleotide tail sequence comprising first and second regions, the nucleotides of the first region being complementary to but in reverse order from the nucleotides of the second region, whereby hybridisation of the first region to the second region results in the quencher group to be sufficiently close to the fluorophore group to quench the fluorophore group.

3. A primer according to claim 2 wherein the tail sequence further comprises a third region having a sequence complementary to a region of the EGFR gene.

4. An isolated EGFR mutation diagnostic primer comprising at least the six nucleotides at the 3′ end of SEQ. ID NO. 6 further comprising a quencher group and a fluorophore group, wherein the quencher group and the fluorophore group are separated by a tail sequence comprising SEQ. ID NO. 20.

5. A primer according to claim 1 wherein the quencher group comprises black hole quencher 1 (BHQ1) and the fluorophore group comprises FAM.

6. A primer according to claim 1 wherein the quencher group comprises black hole quencher 2 (BHQ2) and the fluorophore comprises Cal Red.

7. A kit comprising at least a pair of polynucleotides for combined use, wherein one member of the pair of polynucleotides is an EGFR mutation diagnostic primer and comprises SEQ ID NO: 75 or the sequence complementary to SEQ ID NO: 75 and the other member of the pair of polynucleotides comprises SEQ ID NO: 76 or the sequence complementary to SEQ ID NO: 76, wherein one of the polynucleotides comprises a fluorophore group.

8. A kit comprising at least a set of three polynucleotides for combined use, wherein the set of three polynucleotides comprises one of the following sets of three primer sequences, respectively, or sequences complementary thereto: SEQ. ID NOS. 1, 10 and 15, or SEQ. ID NOS. 2, 10 and 15, or SEQ. ID NOS. 4, 11 and 15, or SEQ. ID NOS. 5, 11 and 15, or SEQ. ID NOS. 6, 12 and 16, or SEQ. ID NOS. 21, 10 and 15, or SEQ. ID NOS. 74, 75 and 76, wherein one of the polynucleotides of each set comprises a fluorophore group.

9. A kit according to claim 7 further comprising nucleotide triphosphates, a polymerisation enzyme and/or a buffer solution.

10. A method of detecting the presence or absence of a mutation in the EGFR gene comprising the steps of:
a) mixing a nucleic acid sample comprising at least a fragment of the EGFR gene with an EGFR mutation diagnostic primer, wherein said primer comprises one of the following primer sequences, or the sequence complimentary to one of the following primer sequences: SEQ. ID NOS. 1, 2, 4, 5, 6, 19, 20, 21 or 75; and
b) detecting hybridisation of the EGFR mutation diagnostic primer to the nucleic acid sample wherein hybridisation indicates the presence or absence of a mutation.

11. A method according to claim 10, wherein step b) comprises carrying out DNA polymerisation using the EGFR mutation diagnostic primer as a first primer and detecting the extension product of polymerisation.

12. A method according to claim 11 wherein step b) comprises the step of mixing the nucleic acid sample and the EGFR mutation diagnostic primer with a second primer which corresponds to a region of the fragment of the EGFR sequence downstream of the region to which the EGFR mutation diagnostic primer is complementary and carrying out PCR on the mixture.

13. A method according to claim 12 wherein the second primer comprises: SEQ. ID NO. 15 and the EGFR mutation diagnostic primer comprises SEQ. ID NOS. 1, 2, 4, 5, or 21; SEQ. ID NO. 16 and the EGFR mutation diagnostic primer comprises SEQ. ID NO. 6; or SEQ. ID NO. 76 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NO. 75.

14. A method according to claim 11 wherein step a) comprises the step of mixing the nucleic acid sample with a pair of a mutation specific primer and a wild-type specific primer, the pair being selected from: SEQ. ID NO: 1 and SEQ. ID NO: 10; SEQ. ID NO: 2 and SEQ. ID NO: 10; SEQ. ID NO: 4 and SEQ. ID NO: 11; SEQ. ID NO: 5 and SEQ. ID NO: 11; SEQ. ID NO: 6 and SEQ. ID NO: 12; SEQ. ID NO: 21 and SEQ. ID NO: 10; or SEQ. ID NO. 74 and SEQ. ID NO. 75.

15. A method according to claim 14 wherein the nucleic acid sample comprises wild-type sequences and mutated sequences and further comprising step c) wherein the number of amplification cycles required to amplify the wild-type sequences to a predetermined quantity is compared with the number of amplification cycles required to amplify the mutated sequences to the predetermined quantity thereby providing an indication of the ratio of the wild type sequences to mutated sequences in the sample.

16. A method according to claim 15 wherein the nucleic acid sample comprises a portion of tumourous tissue and a portion of non-tumourous tissue and wherein step c) further comprises the step of determining the ratio of tumourous tissue to non-tumourous tissue in the sample.

17. A method according to claim 15 or 16 further comprising the step of, prior to step a), enriching the nucleic acid sample to increase the ratio of tumourous tissue to non-tumourous tissue in the sample.

18. A method according to claim 13 wherein step b) comprises detecting if amplification of at least a portion of the EGFR gene occurs.

19. A method according to claim 10 wherein the EGFR mutation diagnostic primer comprises a quencher group and a fluorophore group and wherein step b) comprises exposing the mixture to light of a wavelength to which the fluorophore is responsive in the absence of the quencher group and detecting light at the wavelength emitted by the fluorophore group in the absence of the quencher group.

20. A composition comprising an EGFR mutation diagnostic primer according to claim 1, wherein the EGFR mutation diagnostic primer is not bound to a solid substrate.