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.

1 . An isolated EGFR mutation diagnostic primer comprising one of the following primer sequences, or a sequence complementary to one of the following primer sequences: SEQ. ID NOS. 1, 6 or 75. 
     
     
         2 . A primer according to  claim 1 , wherein the primer 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. 
     
     
         3 . A primer according to  claim 1 , further comprising a quencher group and a fluorophore group. 
     
     
         4 . A primer according to  claim 3 , 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, 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. 
     
     
         5 . A primer according to  claim 4 , wherein the tail sequence further comprises a third region having a sequence complementary to a region of the EGFR gene. 
     
     
         6 . A primer according to  claim 3 , wherein the quencher group comprises black hole quencher 1 (BHQ1) and the fluorophore group comprises FAM. 
     
     
         7 . A primer according to  claim 3 , wherein the quencher group comprises black hole quencher 2 (BHQ2) and the fluorophore comprises Cal Red. 
     
     
         8 . A kit comprising at least a pair of polynucleotides for combined use, wherein
 (a) one member of the pair of polynucleotides is an EGFR mutation diagnostic primer and comprises SEQ. ID NO. 1 or a sequence complementary to SEQ. ID NO. 1, and the other member of the pair of polynucleotides comprises SEQ. ID NO. 15 or a sequence complementary to SEQ. ID NO. 15;   (b) one member of the pair of polynucleotides is an EGFR mutation diagnostic primer and comprises SEQ. ID NO. 6 or a sequence complementary to SEQ. ID NO. 6, and the other member of the pair of polynucleotides comprises SEQ. ID NO. 16 or a sequence complementary to SEQ. ID NO. 16;   (c) one member of the pair of polynucleotides is an EGFR mutation diagnostic primer and comprises SEQ. ID NO. 75 or a sequence complementary to SEQ. ID NO. 75, and the other member of the pair of polynucleotides comprises SEQ. ID NO. 76 or a sequence complementary to SEQ. ID NO. 76.   
     
     
         9 . A kit according to  claim 8 , further comprising nucleotide triphosphates, a polymerisation enzyme and/or a buffer solution. 
     
     
         10 . 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:
 (a) SEQ. ID NOS. 1, 10 and 15, or   (b) SEQ. ID NOS. 6, 12 and 16, or   (c) SEQ. ID NOS. 74, 75 and 76.   
     
     
         11 . A kit according to  claim 10 , further comprising nucleotide triphosphates, a polymerisation enzyme and/or a buffer solution. 
     
     
         12 . 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 at least four or five of the final six nucleotides of one of the following primer sequences: SEQ. ID NOS. 1, 6 or 75, or a sequence complementary thereto; 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.   
     
     
         13 . A method according to  claim 12 , further comprising 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. 
     
     
         14 . A method according to  claim 12 , 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. 
     
     
         15 . A method according to  claim 12 , 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 polynucleotide is complementary and carrying out PCR on the mixture. 
     
     
         16 . A method according to  claim 15 , wherein
 (a) the second primer comprises SEQ. ID NO. 15 and the EGFR mutation diagnostic primer comprises at least four or five of the final six nucleotides of SEQ. ID NO. 1;   (b) the second primer comprises SEQ. ID NO. 16 and the EGFR mutation diagnostic primer comprises at least four or five of the final six nucleotides of SEQ. ID NO. 6;   (c) the second primer comprises SEQ. ID NO. 76 and the EGFR mutation diagnostic primer comprises at least four or five of the final six nucleotides of SEQ. ID NO. 75;   
     
     
         17 . A method according to  claim 12 , wherein 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 nucleotides of: SEQ. ID NO: 1 and SEQ. ID NO: 10; SEQ. ID NO: 6 and SEQ. ID NO: 12; or SEQ. ID NO. 74 and SEQ. ID NO. 75. 
     
     
         18 . A method according to  claim 17 , 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. 
     
     
         19 . A method according to  claim 18 , 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 determining the ratio of tumourous tissue to non-tumourous tissue in the sample. 
     
     
         20 . A method according to  claim 18 , 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. 
     
     
         21 . A method according to  claim 15 , wherein step b) comprises detecting if amplification of at least a portion of the EGFR gene occurs. 
     
     
         22 . A method according to  claim 12 , wherein the EGFR mutation diagnostic primer comprise 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. 
     
     
         23 . 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.